Compounds and methods for conjugation of biomolecules

ABSTRACT

Low-copper click chemistry, 1.3-dipolar cycloadditions, and Staudinger ligations for modifying biomolecules is provided. Compositions, methods, and kits relating to low-copper click chemistry, 1.3-dipolar cycloadditions, and Staudinger ligations are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. Nonprovisional applicationSer. No. 14/000,819, filed Mar. 6, 2014, which application is a 371application of International Application No. PCT/US2012/027285, filedMar. 1, 2012, which claims the benefit of priority to U.S. ProvisionalApplication No. 61/558,148, filed Nov. 10, 2011, and U.S. ProvisionalApplication No. 61/449,396, filed Mar. 4, 2011, which disclosures areherein incorporated by reference in their entirety.

FIELD

This invention relates to click chemistry, 1,3-dipolar cycloadditions,and Staudinger ligations for conjugating biomolecules.

BACKGROUND

Conjugation of biomolecules, such as polynucleotides, proteins, lipids,etc., can be useful for detection, isolation, and/or identification ofbiomolecules. Click chemistry was developed by K. Barry Sharpless as arobust and specific method of ligating two molecules together. See,e.g., Kolb et al. Angew. Chemie Intern. 40(11): 2004-21 (2001). Classicclick reactions typically require Cu(I) ions in order to proceedefficiently. However, Cu(I) ions can have deleterious effects on cellsand biomolecules. Reducing the amount and/or accessibility of Cu(I) ionsused in click reactions could therefore be beneficial for conjugatingbiomolecules.

SUMMARY

In one aspect, the invention provides a compound of the formula:

wherein:

A is a carbon, or A, R₅, and R₆ are absent;

R₁, R₂, R₃, and R₄, are independently selected from hydrogen, halogen,—SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl, arylcarboxamido, alkyl and aryl portions areoptionally substituted one or more times by halogen, —SO₃X, a carboxylicacid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido; ortwo substituents selected from R₁, R₂, R₃, and R₄, wherein each of theat least two substituents are on different carbon atoms together form afused moiety selected from cycloalkyl, heterocycloalkyl, substitutedcycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substitutedaryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, andall of the remaining substituents are independently selected fromhydrogen, halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl; or at least two of the remaining substituentstogether form a fused moiety selected from cycloalkyl, heterocycloalkyl,substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl, and any remaining substituents are independently selectedfrom hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl;

R₅, and R₆, are independently selected from hydrogen, halogen, —SO₃X, acarboxylic acid, a salt of carboxylic acid, CN, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido,alkyl and aryl portions are optionally substituted one or more times byhalogen, —SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl, arylcarboxamido;

at least one substituent selected from R₁, R₂, R₃, R₄, R₅ and R₆comprises X-L-, wherein:

-   -   X is selected from a reporter molecule, a carrier molecule, a        solid phase, a therapeutic molecule such as peptide, a protein,        an antibody, a polysaccharide, a nucleic acid polymer, an ion        complexing moiety, a lipid or a non-biological organic polymer        or polymeric micro or nano particle, that are optionally bound        to one or more additional fluorophores; or    -   X is a reactive group such as carboxylic acid, an activated        ester of carboxylic acid, an amine, a hydrazine, a        haloacetamide, an alkyl halide, an isothiocynate or a maleimide        group; and    -   L is an independently a single covalent bond or L is covalent        linkage having 1-24 non-hydrogen atoms selected from the group        consisting of C, N, O, P and S and composed of any combinations        of single, double, triple or aromatic carbon-carbon bonds,        carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen        bonds, carbon-sulfur bonds, phosphorus-oxygen bonds and        phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,        cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,        substituted alkyl, substituted heteroalkyl, substituted        cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,        substituted aryl, arylalkyl, substituted arylalkyl, and        substituted heteroaryl;

Z is an independently a single covalent bond or Z is covalent linkagehaving 1-10 non-hydrogen atoms selected from the group consisting of C,N, O, P and S and composed of any combinations of single, double, tripleor aromatic carbon-carbon bonds, carbon-nitrogen bonds, carbon-oxygenbonds, and carbon-sulfur bonds in the form of a straight- orbranched-chain alkyl or heteroalkyl chain; and

G is a chemical handle selected from an azide-reactive group, analkyne-reactive group, and a phosphine-reactive group.

In some embodiments, the compound is of the formula (I). In some ofthese, the compound is of the formula:

In others, the compound is of the formula:

wherein

R₂, R₃, R₄, and R₇ to R₁₂ are independently selected from hydrogen,halogen, —SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl, arylcarboxamido, alkyl and aryl portions areoptionally substituted one or more times by halogen, —SO₃X, a carboxylicacid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido; ortwo substituents selected from R₂, R₃, R₄, and R₇ to R₁₂, wherein thetwo substituents are on different carbon atoms together form a fusedmoiety selected from cycloalkyl, heterocycloalkyl, substitutedcycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substitutedaryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, andall of the remaining substituents are independently selected fromhydrogen, halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl; or two of the remaining substituents alsotogether form a fused moiety selected from cycloalkyl, heterocycloalkyl,substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl, and the remaining substituents are independently selectedfrom hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl; or R₇ to R₁₂, whereinthe two substituents are on same carbon atom together form a spirocyclicmoiety selected from alkyl or hetertoalkyl, portions of which arefurther optionally substituted with halogen, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,alkoxy, substituted alkoxy, aryl, heteroaryl, substituted aryl,arylalkyl, substituted arylalkyl, and substituted heteroaryl;

at least one substituent selected from R₁, R₂, R₃, R₄, R₅ and R₆comprises X-L-, wherein:

-   -   X is selected from a reporter molecule, a carrier molecule, a        solid phase, a therapeutic molecule such as peptide, a protein,        an antibody, a polysaccharide, a nucleic acid polymer, an ion        complexing moiety, a lipid or a non-biological organic polymer        or polymeric micro or nano particle, that are optionally bound        to one or more additional fluorophores; or    -   X is a reactive group such as carboxylic acid, an activated        ester of carboxylic acid, an amine, a hydrazine, a        haloacetamide, an alkyl halide, an isothiocynate or a maleimide        group; and    -   L is an independently a single covalent bond or L is covalent        linkage having 1-24 non-hydrogen atoms selected from the group        consisting of C, N, O, P and S and composed of any combinations        of single, double, triple or aromatic carbon-carbon bonds,        carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen        bonds, carbon-sulfur bonds, phosphorus-oxygen bonds and        phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,        cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,        substituted alkyl, substituted heteroalkyl, substituted        cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,        substituted aryl, arylalkyl, substituted arylalkyl, and        substituted heteroaryl; and

G is a chemical handle selected from an azide-reactive group, analkyne-reactive group, and a phosphine-reactive group.

In some embodiments, the compound of the formula (I), (II) or (III) isselected from the group consisting of:

In another aspect, the invention provides a compound of the formula:

wherein:

R₁, R₂, R₃, and R₄ are independently selected from hydrogen, halogen,—SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl, arylcarboxamido, alkyl and aryl portions areoptionally substituted one or more times by halogen, —SO₃X, a carboxylicacid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido; ortwo substituents selected from R₁, R₂, R₃, and R₄ together form a fusedmoiety selected from cycloalkyl, heterocycloalkyl, substitutedcycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substitutedaryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, andthe remaining two substituents also together form a fused moietyselected from cycloalkyl, heterocycloalkyl, substituted cycloalkyl,substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,arylalkyl, substituted arylalkyl, and substituted heteroaryl, or theremaining two substituents are independently selected from hydrogen,halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substitutedalkyl, substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl;

R₅, and R₆, are independently selected from hydrogen, —SO₃X, acarboxylic acid, a salt of carboxylic acid, CN, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido,alkyl and aryl portions are optionally substituted one or more times byhalogen, —SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl, arylcarboxamido; or

at least one substituent selected from R₁, R₂, R₃, R₄, R₅, and R₆comprises X-L-, wherein:

-   -   X is selected from a reporter molecule, a carrier molecule, a        solid phase, or a therapeutic molecule such as peptide, a        protein, an antibody, a polysaccharide, a nucleic acid polymer,        an ion complexing moiety, a lipid or a non-biological organic        polymer or polymeric micro or nano particle, that are optionally        bound to one or more additional fluorophores; or    -   X is a reactive group such as carboxylic acid, an activated        ester of carboxylic acid, an amine, a hydrazine, a        haloacetamide, an alkyl halide, an isothiocynate or a maleimide        group; and    -   L is an independently a single covalent bond or L is covalent        linkage having 1-24 non-hydrogen atoms selected from the group        consisting of C, N, O, P and S and composed of any combinations        of single, double, triple or aromatic carbon-carbon bonds,        carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen        bonds, carbon-sulfur bonds, phosphorus-oxygen bonds and        phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,        cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,        substituted alkyl, substituted heteroalkyl, substituted        cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,        substituted aryl, arylalkyl, substituted arylalkyl, and        substituted heteroaryl;

A is a carbon, and R₅ and R₆ are independently selected from hydrogen,halogen, —SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl, arylcarboxamido, alkyl and aryl portions areoptionally substituted one or more times by halogen, —SO₃X, a carboxylicacid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido or A,R₅, and R₆ are absent;

B is selected from O, S, and NR₇, wherein R₇ is selected from hydrogen,halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substitutedalkyl, substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl and X-L wherein:

-   -   X is selected from a reporter molecule, a carrier molecule, a        solid phase, a therapeutic molecule such as peptide, a protein,        an antibody, a polysaccharide, a nucleic acid polymer, an ion        complexing moiety, a lipid or a non-biological organic polymer        or polymeric micro or nano particle, that are optionally bound        to one or more additional fluorophores; or    -   X is a reactive group such as carboxylic acid, an activated        ester of carboxylic acid, an amine, a hydrazine, a        haloacetamide, an alkyl halide, an isothiocynate or a maleimide        group; and    -   L is an independently a single covalent bond or L is covalent        linkage having 1-24 non-hydrogen atoms selected from the group        consisting of C, N, O, P and S and composed of any combinations        of single, double, triple or aromatic carbon-carbon bonds,        carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen        bonds, carbon-sulfur bonds, phosphorus-oxygen bonds and        phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,        cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,        substituted alkyl, substituted heteroalkyl, substituted        cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,        substituted aryl, arylalkyl, substituted arylalkyl, and        substituted heteroaryl;

Z is an independently a single covalent bond or Z is covalent linkagehaving 1-10 non-hydrogen atoms selected from the group consisting of C,N, O, P and S and composed of any combinations of single, double, tripleor aromatic carbon-carbon bonds, carbon-nitrogen bonds, carbon-oxygenbonds, and carbon-sulfur bonds in the form of a straight- orbranched-chain alkyl or heteroalkyl chain; and

G is a chemical handle selected from an azide-reactive group, analkyne-reactive group, and a phosphine-reactive group.

In another aspect, the invention provides a compound of the formula:

wherein:

A is a carbon, or A, R₅, and R₆ are absent;

R₁, R₂, R₃ and R₄ are independently selected from hydrogen, halogen,—SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl arylcarboxamido, alkyl and aryl portions areoptionally substituted one or more times by halogen, —SO₃X, a carboxylicacid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido;

R₅, and R₆, are independently selected from hydrogen, —SO₃X, acarboxylic acid, a salt of carboxylic acid, CN, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido,alkyl and aryl portions are optionally substituted one or more times byhalogen, —SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl, arylcarboxamido; or two substituents selectedfrom R₁, R₂, R₃, and R₄, wherein the two substituents are on differentcarbon atoms together form a fused moiety selected from cycloalkyl,heterocycloalkyl, substituted cycloalkyl, substituted heterocycloalkyl,aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl,and substituted heteroaryl, and the remaining substituents areindependently selected from hydrogen, halogen, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,alkoxy, substituted alkoxy, aryl, heteroaryl, substituted aryl,arylalkyl, substituted arylalkyl, and substituted heteroaryl; or

at least one substituent selected from R₁, R₂, R₃, R₄, R₅, and R₆comprises X-L-, wherein:

-   -   X is selected from a reporter molecule, a carrier molecule, a        solid phase, or a therapeutic molecule such as peptide, a        protein, an antibody, a polysaccharide, a nucleic acid polymer,        an ion complexing moiety, a lipid or a non-biological organic        polymer or polymeric micro or nano particle, that are optionally        bound to one or more additional fluorophores; or    -   X is a reactive group such as carboxylic acid, an activated        ester of carboxylic acid, an amine, a hydrazine, a        haloacetamide, an alkyl halide, an isothiocynate or a maleimide        group; and    -   L is an independently a single covalent bond or L is covalent        linkage having 1-24 non-hydrogen atoms selected from the group        consisting of C, N, O, P and S and composed of any combinations        of single, double, triple or aromatic carbon-carbon bonds,        carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen        bonds, carbon-sulfur bonds, phosphorus-oxygen bonds and        phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,        cycloalkyl, heterocycloalkyl, substituted alkyl, substituted        heteroalkyl, substituted cycloalkyl, substituted        heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,        substituted arylalkyl, and substituted heteroaryl;

B, C, D and E are selected from O, S, and N where N is furthersubstituted with either R₇, R₈ or R₉, wherein R₇, R₈ or R₉ are selectedfrom hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl,substituted heteroaryl and X-L wherein:

-   -   X is selected from a reporter molecule, a carrier molecule, a        solid phase, or a therapeutic molecule such as peptide, a        protein, an antibody, a polysaccharide, a nucleic acid polymer,        an ion complexing moiety, a lipid or a non-biological organic        polymer or polymeric micro or nano particle, that are optionally        bound to one or more additional fluorophores; or    -   X is a reactive group such as carboxylic acid, an activated        ester of carboxylic acid, an amine, a hydrazine, a        haloacetamide, an alkyl halide, an isothiocynate or a maleimide        group; and    -   L is an independently a single covalent bond or L is covalent        linkage having 1-24 non-hydrogen atoms selected from the group        consisting of C, N, O, P and S and composed of any combinations        of single, double, triple or aromatic carbon-carbon bonds,        carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen        bonds, carbon-sulfur bonds, phosphorus-oxygen bonds and        phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,        cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,        substituted alkyl, substituted heteroalkyl, substituted        cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,        substituted aryl, arylalkyl, substituted arylalkyl, and        substituted heteroaryl;

Z is an independently a single covalent bond or Z is covalent linkagehaving 1-10 non-hydrogen atoms selected from the group consisting of C,N, O, P and S and composed of any combinations of single, double, tripleor aromatic carbon-carbon bonds, carbon-nitrogen bonds, carbon-oxygenbonds, and carbon-sulfur bonds in the form of a straight- orbranched-chain alkyl or heteroalkyl having a chain length of 1-10 atoms,or is absent; and

G is a chemical handle selected from an azide-reactive group, analkyne-reactive group, and a phosphine-reactive group.

In some embodiments, the compound is of formula (VI), and R₁, R₂, R₃,and R₄, are independently selected from hydrogen, halogen, —SO₃X, acarboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino,alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, llkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido,alkyl and aryl portions are optionally substituted one or more times byhalogen, —SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,substituted heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted aryl,arylalkyl, substituted arylalkyl, and substituted heteroaryl,arylcarboxamido; or R₁ and R₂, R₂ and R₃, R₃ and R₄ together form afused moiety selected from cycloalkyl, heterocycloalkyl, substitutedcycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substitutedaryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, andR₅ and R₆ are independently selected from hydrogen, halogen, alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl.

One embodiment is the compound:

In another aspect, the invention provides a compound of the formula:

wherein:

A is a carbon, or A, R₅, and R₆ are absent;

R₁, R₂, and R₃ are independently selected from hydrogen, halogen, —SO₃X,a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl,amino, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substitutedalkyl, substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido,alkyl and aryl portions are optionally substituted one or more times byhalogen, —SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,substituted heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted aryl,arylalkyl, substituted arylalkyl, and substituted heteroaryl,arylcarboxamido;

R₅, and R₆, are independently selected from hydrogen, halogen, —SO₃X, acarboxylic acid, a salt of carboxylic acid, CN, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido,alkyl and aryl portions are optionally substituted one or more times byhalogen, —SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl, arylcarboxamido; or

two substituents selected from R₁, R₂, R₃, R₅, and R₆, wherein the twosubstituents are on different carbon atoms, together form a fused moietyselected from cycloalkyl, heterocycloalkyl, substituted cycloalkyl,substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,arylalkyl, substituted arylalkyl, and substituted heteroaryl, and theremaining substituents are selected from hydrogen, halogen, alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl;

at least one substituent selected from R₁, R₂, R₃, R₅, and R₆ comprisesX-L-, wherein:

-   -   X is selected from a reporter molecule, a carrier molecule, a        solid phase, or a therapeutic molecule such as peptide, a        protein, an antibody, a polysaccharide, a nucleic acid polymer,        an ion complexing moiety, a lipid or a non-biological organic        polymer or polymeric micro or nano particle, that are optionally        bound to one or more additional fluorophores; or    -   X is a reactive group such as carboxylic acid, an activated        ester of carboxylic acid, an amine, a hydrazine, a        haloacetamide, an alkyl halide, an isothiocynate or a maleimide        group; and    -   L is an independently a single covalent bond or L is covalent        linkage having 1-24 non-hydrogen atoms selected from the group        consisting of C, N, O, P and S and composed of any combinations        of single, double, triple or aromatic carbon-carbon bonds,        carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen        bonds, carbon-sulfur bonds, phosphorus-oxygen bonds and        phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,        cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,        substituted alkyl, substituted heteroalkyl, substituted        cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,        substituted aryl, arylalkyl, substituted arylalkyl, and        substituted heteroaryl;

Z is an independently a single covalent bond or Z is covalent linkagehaving 1-10 non-hydrogen atoms selected from the group consisting of C,N, O, P and S and composed of any combinations of single, double, tripleor aromatic carbon-carbon bonds, carbon-nitrogen bonds, carbon-oxygenbonds, and carbon-sulfur bonds in the form of a straight- orbranched-chain alkyl or heteroalkyl having a chain length of 1-10 atoms,or is absent; and

G is a chemical handle selected from an azide-reactive group, analkyne-reactive group, and a phosphine-reactive group.

In another aspect, the invention provides a compound of the formula:

wherein:

A is a carbon, or A, R₅, and R₆ are absent;

R₁, and R₂ are independently selected from hydrogen, halogen, —SO₃X, acarboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino,alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl,substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido,alkyl and aryl portions are optionally substituted one or more times byhalogen, —SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,substituted heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted aryl,arylalkyl, substituted arylalkyl, and substituted heteroaryl,arylcarboxamido;

R₅, and R₆, are independently selected from hydrogen, —SO₃X, acarboxylic acid, a salt of carboxylic acid, CN, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido,alkyl and aryl portions are optionally substituted one or more times byhalogen, —SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl, arylcarboxamido; or

at least one substituent selected from R₁, R₂, R₅, and R₆ comprisesX-L-, wherein:

-   -   X is selected from a reporter molecule, a carrier molecule, a        solid phase, or a therapeutic molecule such as peptide, a        protein, an antibody, a polysaccharide, a nucleic acid polymer,        an ion complexing moiety, a lipid or a non-biological organic        polymer or polymeric micro or nano particle that are optionally        bound to one or more additional fluorophores; or    -   X is a reactive group such as carboxylic acid, an activated        ester of carboxylic acid, an amine, a hydrazine, a        haloacetamide, an alkyl halide, an isothiocynate or a maleimide        group; and    -   L is an independently a single covalent bond or L is covalent        linkage having 1-24 non-hydrogen atoms selected from the group        consisting of C, N, O, P and S and composed of any combinations        of single, double, triple or aromatic carbon-carbon bonds,        carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen        bonds, carbon-sulfur bonds, phosphorus-oxygen bonds and        phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,        cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,        substituted alkyl, substituted heteroalkyl, substituted        cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,        substituted aryl, arylalkyl, substituted arylalkyl, and        substituted heteroaryl;

Z is an independently a single covalent bond or Z is covalent linkagehaving 1-10 non-hydrogen atoms selected from the group consisting of C,N, O, P and S and composed of any combinations of single, double, tripleor aromatic carbon-carbon bonds, carbon-nitrogen bonds, carbon-oxygenbonds, and carbon-sulfur bonds in the form of a straight- orbranched-chain alkyl or heteroalkyl having a chain length of 1-10 atoms,or is absent; and

G is a chemical handle selected from an azide-reactive group, analkyne-reactive group, and a phosphine-reactive group.

In another aspect, the invention provides a compound of the formula:

wherein:

A is a carbon, or A, R₅, and R₆ are absent;

m and n is an integer between 1 and 4;

B is O or S and R₃ and R₄ are absent or N and R₃ or R₄ is absent;

R₇ is selected from hydrogen, alkyl, heteroalkyl, substituted alkyl, andsubstituted heteroalkyl;

R₁, R₂, R₃, R₄, each R′, and each R″ are independently selected fromhydrogen, halogen, —SO₃X, a carboxylic acid, a salt of carboxylic acid,CN, nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl, arylcarboxamido, alkyl and aryl portions areoptionally substituted one or more times by halogen, —SO₃X, a carboxylicacid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido; ortwo substituents selected from R₁, R₂, R₃, R₄, an R′, and an R″, whereinthe two substituents are on different carbon atoms together form a fusedmoiety selected from cycloalkyl, heterocycloalkyl, substitutedcycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substitutedaryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, andall of the remaining substituents are independently selected fromhydrogen, halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl; or two of the remaining substituents alsotogether form a fused moiety selected from cycloalkyl, heterocycloalkyl,substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl, and the remaining substituents are independently selectedfrom hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl;

wherein, R₅, and R₆, are independently selected from hydrogen, —SO₃X, acarboxylic acid, a salt of carboxylic acid, CN, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido,alkyl and aryl portions are optionally substituted one or more times byhalogen, —SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl, arylcarboxamido; or

at least one substituent selected from R₁, R₂, R₃, R₄, R₅, R₆, an R′,and an R″ comprises X-L-, wherein:

-   -   X is selected from a reporter molecule, a carrier molecule, a        solid phase, or a therapeutic molecule such as peptide, a        protein, an antibody, a polysaccharide, a nucleic acid polymer,        an ion complexing moiety, a lipid or a non-biological organic        polymer or polymeric micro or nano particle, that are optionally        bound to one or more additional fluorophores; or    -   X is a reactive group such as carboxylic acid, an activated        ester of carboxylic acid, an amine, a hydrazine, a        haloacetamide, an alkyl halide, an isothiocynate or a maleimide        group; and    -   L is an independently a single covalent bond or L is covalent        linkage having 1-24 non-hydrogen atoms selected from the group        consisting of C, N, O, P and S and composed of any combinations        of single, double, triple or aromatic carbon-carbon bonds,        carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen        bonds, carbon-sulfur bonds, phosphorus-oxygen bonds and        phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,        cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,        substituted alkyl, substituted heteroalkyl, substituted        cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,        substituted aryl, arylalkyl, substituted arylalkyl, and        substituted heteroaryl;

Z is an independently a single covalent bond or Z is covalent linkagehaving 1-10 non-hydrogen atoms selected from the group consisting of C,N, O, P and S and composed of any combinations of single, double, tripleor aromatic carbon-carbon bonds, carbon-nitrogen bonds, carbon-oxygenbonds, and carbon-sulfur bonds in the form of a straight- orbranched-chain alkyl or heteroalkyl having a chain length of 1-10 atoms,or is absent; and

G is a chemical handle selected from an azide-reactive group, analkyne-reactive group, and a phosphine-reactive group.

In some embodiments of the compound of the formula (I), (II), (III),(IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX), (X), (XI) or (XII),all but one of the R substituents are H.

In some embodiments of the compound of the formula (I), (II), (III),(IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX), (X), (XI) or (XII),L is an alkyl group having a chain length of 0 to 15 atoms. In some ofthese, L is an alkyl group having a chain length of 0 to 5 atoms. Inothers, L is —NH—(CH₂)_(n)—NH—C(O)—, wherein n is 1 to 12. In others,the compound is:

In some embodiments of the compound of the formula (I), (II), (III),(IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX), (X), (XI) or (XII),the reporter molecule comprises a chromophore, fluorophore, fluorescentprotein, phosphorescent dye, tandem dye, particle, hapten, enzyme, orradioisotope. In some of these, the fluorophore is a xanthene, coumarin,cyanine, pyrene, oxazine, borapolyazaindacene, or carbopyranine. Inothers, the enzyme is horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or beta-lactamase. In others, the particle is asemiconductor nanocrystal.

In some embodiments of the compound of the formula (I), (II), (III),(IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX), (X), (XI) or (XII),the carrier molecule is an amino acid, peptide, protein, polysaccharide,nucleoside, nucleotide, oligonucleotide, nucleic acid, hapten, psoralen,drug, hormone, lipid, lipid assembly, tyramine, synthetic polymer,polymeric microparticle, biological cell, cellular component, ionchelating moiety, enzymatic substrate, or virus.

In some embodiments of the compound of the formula (I), (II), (III),(IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX), (X), (XI) or (XII),the carrier molecule is an antibody, antibody fragment, antigen, avidin,streptavidin, biotin, dextran, IgG binding protein, fluorescent protein,agarose, or non-biological microparticle.

In some embodiments of the compound of the formula (I), (II), (III),(IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX), (X), (XI) or (XII),the solid support is an aerogel, hydrogel, resin, bead, biochip,microfluidic chip, silicon chip, multi-well plate, membrane, conductingmetal, nonconducting metal, glass, or magnetic support.

In some embodiments of the compound of the formula (I), (II), (III),(IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX), (X), (XI) or (XII),the solid support is a silica gel, polymeric membrane, particle,derivatized plastic film, glass bead, cotton, plastic bead, alumina gel,polysaccharide, poly(acrylate), polystyrene, poly(acrylamide), polyol,agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen,amylopectin, mannan, inulin, nitrocellulose, diazocellulose,polyvinylchloride, polypropylene, polyethylene, nylon, latex bead,magnetic bead, paramagnetic bead, superparamagnetic bead, or starch.

In some embodiments of the compound of the formula (I), (II), (III),(IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX), (X), (XI) or (XII),the therapeutic molecule is taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine,propranolol, puromycin, or analogs or homologs thereof.

In some embodiments of the compound of the formula (I), (II), (III),(IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX), (X), (XI) or (XII),the therapeutic molecule is an antimetabolite, alkylating agent,anthracycline, antibiotic, or anti-mitotic agent.

In some embodiments of the compound of the formula (I), (II), (III),(IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX), (X), (XI) or (XII),the therapeutic molecule is abrin, ricin A, pseudomonas exotoxin,diphtheria toxin, tumor necrosis factor, γ-interferon, α-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, interleukin-1, interleukin-2, interleukin-6, granulocytemacrophage colony stimulating factor, or granulocyte colony stimulatingfactor.

In some embodiments of the compound of the formula (I), (II), (III),(IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX), (X), (XI) or (XII),G is an alkyne-reactive group. In some of these, the alkyne-reactivegroup is an azide.

In some embodiments of the compound of the formula (I), (II), (III),(IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX), (X), (XI) or (XII),G is an azide-reactive group. In some of these, the azide-reactive groupis a terminal alkyne.

In another aspect, the invention provides a method of modifying abiomolecule comprising the step of reacting in a solution a biomoleculecomprising an azide reactive moiety with a compound of the formula (I),(II), (III), (IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX), (X),(XI) or (XII), wherein the therapeutic molecule is an antimetabolite,alkylating agent, anthracycline, antibiotic, or anti-mitotic agent toprovide a modified biomolecule.

In some embodiments, the azide reactive moiety comprises a terminalalkyne, an activated alkyne, or triarylphosphine.

In another aspect, the invention provides a method of modifying abiomolecule comprising the step of reacting in a solution a biomoleculecomprising an alkyne reactive moiety with a compound of the formula (I),(II), (III), (IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX), (X),(XI) or (XII), wherein G is an alkyne reactive moiety to provide amodified biomolecule.

In some embodiments, the alkyne reactive moiety comprises an azide.

In some embodiments, the biomolecule is a nucleic acid, oligonucleotide,protein, peptide, carbohydrate, polysaccharide, glycoprotein, lipid,hormone, drug, or prodrug.

In some embodiments, the solution further comprises copper ions. In someof these, the solution further comprises at least one reducing agent. Insome of these, the at least one reducing agent is ascorbate,Tris(2-Carboxyethyl) Phosphine (TCEP), TCP (2,4,6-trichlorophenol),NADH, NADPH, thiosulfate, 2-mercaptoethanol, dithiothreitol,glutathione, cysteine, metallic copper, quinone, hydroquinone, vitaminK1, Fe2+, Co2+, or an applied electric potential. In some of these, theat least one reducing agent is ascorbate.

In some of these, the solution further comprises a copper chelator. Insome of these, the copper chelator is a copper I chelator. In some ofthese, the copper chelator I is a compound of formula:

wherein X, Y, and Z each independently have the formula:

wherein:

R₁, R₂, R₃, R₄ and R₅ are independently selected from hydrogen, halogen,—SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, alkyl, heteroalkyl, alkoxy, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl,substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl, arylcarboxamido, alkyl and aryl portions are optionallysubstituted one or more times by halogen, —SO₃X, a carboxylic acid, asalt of carboxylic acid, CN, nitro, hydroxyl, amino, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido; and

B, C, D, and E are C or N; and

L₁, L₂, and L₃ are covalent linkage having 1-5 non-hydrogen atomsselected from the group consisting of C, N, O, P and S and composed ofany combinations of single, double, triple or aromatic carbon-carbonbonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygenbonds, carbon-sulfur bonds, and phosphorus-nitrogen bonds in the form ofalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, substitutedalkoxy, substituted alkyl, substituted heteroalkyl, substitutedcycloalkyl, and substituted heterocycloalkyl.

In some of these, R₁, R₂, R₃, R₄ and R₅ for at least one substituentselected from X, Y, and Z are each H. In some of these, R₁, R₂, R₃, R₄and R₅ for each of X, Y, and Z are each H.

In some of these, L₁, L₂ and L₃ are each alkyl groups having a chainlength of 1-5 atoms.

In some of these, L₁, L₂ and L₃ are each —CH₂CH₂—.

In others, the copper chelator isN,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), EDTA,neocuproine, N-(2-acetamido)iminodiacetic acid (ADA),pyridine-2,6-dicarboxylic acid (PDA), S-carboxymethyl-L-cysteine (SCMC),1,10 phenanthroline, or a derivative thereof, trientine, glutathione,histidine, polyhistidine, tris-(hydroxypropyltriazolylmethyl)amine(THPTA), or tetra-ethylenepolyamine (TEPA).

In others, the copper chelator is 1,10 phenanthroline,bathophenanthroline disulfonic acid (4,7-diphenyl-1,10-phenanthrolinedisulfonic acid), or bathocuproine disulfonic acid(2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate).

In another aspect, the provides a kit comprising a compound of theformula (I), (II), (III), (IV), (XIII), (V), (VI), (VII), (VIII), (XIV),(IX), (X), (XI) or (XII).

In some embodiments, the kit further comprises a copper ion source.

In some embodiments, the kit further comprises at least one reducingagent. In some of these, the at least one reducing agent is ascorbate,Tris(2-Carboxyethyl) Phosphine (TCEP), TCP (2,4,6-trichlorophenol),NADH, NADPH, thiosulfate, 2-mercaptoethanol, dithiothreitol,glutathione, cysteine, metallic copper, quinone, hydroquinone, vitaminK1, Fe2+, Co2+, or an applied electric potential. In others, the atleast one reducing agent is ascorbate.

In some embodiments, the kit further comprises a copper chelator. Insome of these, the copper chelator is a copper I chelator. In some ofthese, the copper chelator I is a compound of formula:

wherein X, Y, and Z each independently have the formula:

wherein:

R₁, R₂, R₃, R₄ and R₅ are independently selected from hydrogen, halogen,—SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, alkylthio, alkanoylamino, alkylaminocarbonyl,arylcarboxamido, alkyl aryl portions are optionally substituted one ormore times by halogen, —SO₃X, a carboxylic acid, a salt of carboxylicacid, CN, nitro, hydroxyl, amino, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl, arylcarboxamido; and

B, C, D, and E are C or N; and

L₁, L₂, and L₃ are covalent linkage having 1-5 non-hydrogen atomsselected from the group consisting of C, N, O, P and S and composed ofany combinations of single, double, triple or aromatic carbon-carbonbonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygenbonds, carbon-sulfur bonds, and phosphorus-nitrogen bonds in the form ofalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, substitutedalkoxy, substituted alkyl, substituted heteroalkyl, substitutedcycloalkyl, and substituted heterocycloalkyl.

In some of these, R₁, R₂, R₃, R₄ and R₅ for at least one substituentselected from X, Y, and Z are each H. In some of these, R₁, R₂, R₃, R₄and R₅ for each of X, Y, and Z are each H.

In some of these, L₁, L₂ and L₃ are each alkyl groups having a chainlength of 1-5 atoms. In some of these, L₁, L₂ and L₃ are each —CH₂CH₂—.

In others, the copper chelator isN,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), EDTA,neocuproine, N-(2-acetamido)iminodiacetic acid (ADA),pyridine-2,6-dicarboxylic acid (PDA), S-carboxymethyl-L-cysteine (SCMC),1,10 phenanthroline, or a derivative thereof, trientine, glutathione,histidine, polyhistidine, tris-(hydroxypropyltriazolylmethyl)amine(THPTA), or tetra-ethylenepolyamine (TEPA).

In others, the copper chelator is 1,10 phenanthroline,bathophenanthroline disulfonic acid (4,7-diphenyl-1,10-phenanthrolinedisulfonic acid), or bathocuproine disulfonic acid(2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate).

In another aspect, the invention provides a compound selected from thegroup consisting of:

In another aspect, the invention provides a composition comprising acompound of the formula (I), (II), (III), (IV), (XIII), (V), (VI),(VII), (VIII), (XIV), (IX), (X), (XI) or (XII); and a copper chelator Iof the formula (XV).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a reaction scheme for (A) the synthesis of tert-butyl(2-(6-(azidomethyl)nicotinamido)ethyl) carbamate (6), and (B)fluorescent-labeling of compound (6), as described in Example 1.

FIG. 2 shows a reaction scheme for the synthesis of tert-butyl(2-(6-((prop-2-yn-1-yloxy)methyl)nicotinamido) ethyl)carbamate (12), andfluorescent labeling of compound (12), as described in Example 1.

FIG. 3 shows rates of click reactions between reagents (A) QSY pAzideand Oregon Green® alkyne, and (B) QSY Azide and Oregon Green® alkyne, atvarious concentrations of Cu, as described in Example 2.

FIG. 4 shows the effect of increasing concentrations of Cu²⁺, andincreasing concentrations of Cu²⁺ in the presence of copper chelatorTHPTA on the click reaction between Oregon Green® alkyne and QSY Azideor QSY pAzide, as described in Example 2.

FIG. 5 shows shows the results of click reactions between acoumarin-alkyne (16) and various azides in the presence of (A) 125 μMCu, or (B) 31 μM Cu, with or without THPTA at a ratio of 4:1 (THPTA:Cu),as described in Example 4.

FIG. 6 shows the stability of GFP in the presence of (A) variousconcentrations of Cu(II), and (B) various concentrations of Cu(II) withsodium ascorbate, as described in Example 5.

FIG. 7 shows (A) the stability of GFP in the presence of Cu(II), with orwithout ascorbate, and with or without THPTA, and (B) the rate of aclick reaction between Oregon Green® alkyne and QSY azide under the sameconcentrations shown in (A), as described in Example 5.

FIG. 8 shows Click Labeling of RNA with EU in presence of GPF withAF647-picolyl azide or AF647 azide with or without THPTA as ligand asdescribed in Example 6.

FIG. 9 shows Click Labeling of RNA with EU in presence of GPF withAF647-picolyl azide or AF647 azide with THPTA as ligand with variousmolar ratio of Cu:THPTA. as described in Example 6.

FIG. 10 shows Graphical representation of GFP stability and progressionof click reaction at 2 mM copper as described in Example 6.

FIG. 11 shows Graphical representation of click reaction rate with orwithout THPTA at different molar ratios of Cu:THPTA as described inExample 6

FIG. 12 shows Graphical representation of GFP stability and progressionof click reaction at 200 μM copper

FIG. 13 shows Click Labeling of HPG with AF647-picolyl azide or AF647azide in presence of GPF with THPTA as described in Example 7.

FIG. 14 shows click Labeling of HPG with AF647-picolyl azide or AF647azide in presence of GPF with THPTA as ligand with various molar ratioof Cu:THPTA as described in Example 7.

FIG. 15 shows graphical representation of GFP stability and progressionof click reaction with HPG at 2 mM copper as described in Example 7.

FIG. 16 shows shows Click Labeling of HPG with AF647-picolyl azidefollowed by phalloidin staining in presence of GPF with THPTA asdescribed in Example 8.

FIG. 17 shows click Labeling of HPG with AF647-picolyl azide or AF647azide in presence of GPF with THPTA as ligand with various molar ratioof Cu:THPTA as described in Example 8.

FIG. 18 shows a SDS-PAGE gel GalNAz-labeled monoclonal anti-TSH clickreacted with either DIBO or click reacted THPTA/Picolyl, where in thelatter the THPTA is varied with respect to the Cu concentration.

FIG. 19 shows the ligation of a picolyl azide derivative bearing acarboxylate terminal onto recombinant proteins expressed on the surfaceof living mammalian cells, as described in Example 10.

FIG. 20 shows the HPLC analysis of ^(W37V)Lp1A-catalyzed ligation ofpicolyl azide (5-(6-(azidomethyl)nicotinamido)pentanoic acid) onto LAPpeptide, as described in Example 10.

FIG. 21 shows the chelation-assisted copper(I) catalyzed azide-alkynecycloaddition for tagging of Alexa Fluor® 647 onto neurexin in livingHEK cells. Negative controls are shown with ATP omitted (second column)or wild-type Lp1A in place of ^(W37V)Lp1A (third column). H2B-YFP is thenuclear-localized YFP transfection marker, as described in Example 10.

FIG. 22 shows the chelation-assisted copper(I) catalyzed azide-alkynecycloaddition for tagging of Alexa Fluor® 647 onto neuroligin-1 inliving hippocampal neurons, as described in Example 10.

DETAILED DESCRIPTION

The present invention has utility in the study of biomolecules, both invivo and in vitro.

The present invention provides compositions, methods, and kits for thelabeling, detecting, isolating and/or analysis of biomolecules modifiedby attachment of chemical handles.

Definitions and Abbreviations

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a ligand” includes aplurality of ligands and reference to “an antibody” includes a pluralityof antibodies and the like.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds described herein may be prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose andappropriate method for a particular situation. See, generally, Furnisset al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY5^(TH) ED., Longman Scientific and Technical Ltd., Essex, 1991, pp.809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

The compounds disclosed herein may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are intended to beencompassed within the scope of the present invention.

Where a disclosed compound includes a conjugated ring system, resonancestabilization may permit a formal electronic charge to be distributedover the entire molecule. While a particular charge may be depicted aslocalized on a particular ring system, or a particular heteroatom, it iscommonly understood that a comparable resonance structure can be drawnin which the charge may be formally localized on an alternative portionof the compound.

Selected compounds having a formal electronic charge may be shownwithout an appropriate biologically compatible counterion. Such acounterion serves to balance the positive or negative charge present onthe compound. As used herein, a substance that is biologicallycompatible is not toxic as used, and does not have a substantiallydeleterious effect on biomolecules. Examples of negatively chargedcounterions include, among others, chloride, bromide, iodide, sulfate,alkanesulfonate, arylsulfonate, phosphate, perchlorate,tetrafluoroborate, tetraarylboride, nitrate and anions of aromatic oraliphatic carboxylic acids. Preferred counterions may include chloride,iodide, perchlorate and various sulfonates. Examples of positivelycharged counterions include, among others, alkali metal, or alkalineearth metal ions, ammonium, or alkylammonium ions.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent (“alkenyl”)and multivalent radicals, having the number of carbon atoms designated(i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include derivatives ofalkyl, such as those defined below, including heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, and substituted heterocycloalkyl. Alkyl groupsthat are limited to hydrocarbon groups are termed “homoalkyl”.

In some embodiments, an alkyl group contains between 1 and 25 carbons,between 1 and 20 carbons (i.e., C₁ to C₂₀ alkyl), between 1 and 15carbons (i.e., C₁ to Cis alkyl), between 1 and 10 carbons (i.e., C₁ toC₁₀ alkyl), or between 1 and 8 carbons (i.e., C₁ to C₈ alkyl). Straight,branched or cyclic hydrocarbon chains having eight or fewer carbon atomsmay also be referred to herein as “lower alkyl”. In addition, the term“alkyl” as used herein may further include one or more substitutions atone or more carbon atoms of the hydrocarbon chain fragment.

The term “carboxyalkyl” as used herein refers to a straight orbranched-chain alkyl including cycloalkyl comprising at least one —COOHsubstituent. The terms “alkoxy,” “alkylamino” and “alkylthio” (orthioalkoxy) are used in their conventional sense, and refer toheteroalkyl groups attached to the remainder of the molecule via anoxygen atom, an amino group, or a sulfur atom, respectively.

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic alkyl, or combinations thereof, with an acyl radical onat least one terminus of the alkyl. An “acyl radical” is a group derivedfrom a carboxylic acid by removing the —OH moiety therefrom.

The term “amino” or “amine group” refers to the group —NR′R′ where R′and R″ are independently selected from hydrogen, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl. In a primary amine group, both R and R′ arehydrogen, whereas in a secondary amine group, either, but not both, R′or R′ is hydrogen. In a tertiary amine group, neither R′ nor R″ is ahydrogen. A substituted amine is an amine group wherein R′ and/or R″ isother than hydrogen. In addition, the terms “amine” and “amino” caninclude protonated and quaternized versions of nitrogen, comprising thegroup —NRR′R″ and its biologically compatible anionic counterions.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), phosphorus (P), silicon (Si), and selenium (Se).

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, P, S, and Se and wherein the nitrogen,phosphorous, sulfur, and selenium atoms are optionally oxidized, and thenitrogen heteroatom is optionally be quaternized. The heteroatom(s) O,N, P, S, Si, and Se may be placed at any interior position of theheteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Examples include, but are notlimited to, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatomsmay be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃. Similarly, the term “heteroalkylene” by itself or aspart of another substituent means a divalent radical derived fromheteroalkyl, as exemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic moiety that can be a single ring or multiple rings (preferablyfrom 1 to 3 rings), which are fused together or linked covalently. Insome embodiments, an aryl group contains twenty or fewer carbon atoms,e.g., phenyl, naphthyl, biphenyl, and anthracenyl. One or more carbonatoms of the aryl group may also be substituted with, e.g., alkyl; aryl;heteroaryl; a halogen; nitro; cyano; hydroxyl, alkoxyl or aryloxyl; thioor mercapto, alkyl-, or arylthio; amino, alkylamino, arylamino,dialkyl-, diaryl-, or arylalkylamino; aminocarbonyl, alkylaminocarbonyl,arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, orarylalkylaminocarbonyl; carboxyl, or alkyl- or aryloxycarbonyl;aldehyde; aryl- or alkylcarbonyl; iminyl, or aryl- or alkyliminyl;sulfo; alkyl- or alkylcarbonyl; sulfo; alkyl- or arylsufonyl;hydroximinyl, or aryl- or alkoximinyl. In addition, two or more alkyl orheteroalkyl substituents of an aryl group may be combined to form fusedaryl-alkyl or aryl-heteroalkyl ring systems (e.g., tetrahydronaphthyl).Substituents including heterocyclic groups (e.g., heteroaryloxy, andheteroaralkylthio) are defined by analogy to the above-described terms.

The term “heteroaryl” refers to aryl groups (or rings) that contain fromone to four heteroatoms selected from N, O, S, and Se, wherein thenitrogen, sulfur, and selenium atoms are optionally oxidized, and thenitrogen atom(s) are optionally quaternized. A heteroaryl group can beattached to the remainder of the molecule through a heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for aryl and heteroaryl ring systems are selected from thegroup of acceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl,”“heteroaryl,” etc.) includes both substituted and unsubstituted forms ofthe indicated radical. Nonlimiting exemplary substituents for each typeof radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound includes morethan one R group, for example, each of the R groups is independentlyselected as are each R′, R″, R′″ and R″″ groups when more than one ofthese groups is present. When R′ and R″ are attached to the samenitrogen atom, they can be combined with the nitrogen atom to form a 5-,6-, or 7-membered ring. For example, —NR′R″ is meant to include, but notbe limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR, ═N—OR′, —NR′R″, —SR, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, ina number ranging from zero to the total number of open valences on thearomatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. In the schemes thatfollow, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

The term “chain length,” as used herein, refers to the smallest numberof carbon and/or heteroatoms between two substituents. As a nonlimitingexample, the chain length between X and Y in the moleculeX—(CH₂)₃—CH(CH₂CH₃)—NH—Y is 5.

The term “activated alkyne,” as used herein, refers to a cyclooctynethat selectively reacts with an azide on another molecule to form acovalent chemical bond between the activated alkyne group and the alkynereactive group. Activated alkynes include, but are not limited to,cyclooctynes and difluorocyclooctynes, described, e.g., in Agard et al.,J. Am. Chem. Soc., 2004, 126 (46):15046-15047; dibenzocyclooctynes,described, e.g., in Boon et al., WO2009/067663 A1 (2009); andaza-dibenzocyclooctynes, described, e.g., in Debets et al., Chem. Comm.,2010, 46:97-99. These dibenzocyclooctynes (including theaza-dibenzocyclooctynes) described above are collectively referred toherein as cyclooctyne groups.

The term “affinity,” as used herein, refers to the strength of thebinding interaction of two molecules, such as an antibody and anantigen, or a positively charged moiety and a negatively charged moiety.For bivalent molecules such as antibodies, affinity is typically definedas the binding strength of one binding domain for the antigen, e.g. oneFab fragment for the antigen. The binding strength of both bindingdomains together for the antigen is referred to as “avidity”. As usedherein “high affinity” refers to a ligand that binds to an antibodyhaving an affinity constant (K_(a)) greater than 10⁴ M⁻¹, typically10⁵-10¹¹ M⁻¹; as determined by inhibition ELISA or an equivalentaffinity determined by comparable techniques such as, for example,Scatchard plots or using K_(d)/dissociation constant, which is thereciprocal of the K_(a).

The term “alkyne reactive,” as used herein, refers to a chemical moietythat selectively reacts with an alkyne, such as a terminal alkyne or anactivated alkyne, on another molecule to form a covalent chemical bondbetween the alkyne modified group and the alkyne reactive group.Examples of alkyne-reactive groups include, but are not limited to,azide and nitrones. “Alkyne-reactive” can also refer to a molecule thatcontains a chemical moiety that selectively reacts with an alkyne group.

The term “antibody,” as used herein, refers to a protein of theimmunoglobulin (Ig) superfamily that binds noncovalently to certainsubstances (e.g. antigens and immunogens) to form an antibody-antigencomplex. Antibodies can be polyclonal or monoclonal. Antibodies can alsobe chimeric, humanized, or human antibodies. It is understood that theterm “antibody” as used herein includes within its scope any of thevarious classes or sub-classes of immunoglobulin derived from any of theanimals conventionally used, or from human.

The term “antibody fragments,” as used herein, refers to fragments ofantibodies that retain the principal selective binding characteristicsof the whole antibody. Nonlimiting exemplary antibody fragment includeFab, Fab′, F(ab′)₂, Fv, and single-chain Fv (scFv). Further nonlimitingexemplary antibody fragments include (i) the Fd fragment, consisting ofthe VH and CH1 domains; (ii) the dAb fragment (Ward, et al., Nature 341,544 (1989)), which consists of a VH domain; and (iii) isolated CDRregions. In addition, arbitrary fragments can be made using recombinanttechnology that retains antigen-recognition characteristics.

The term “antigen,” as used herein, refers to a molecule or molecules towhich an antibody binds selectively. An antigen may comprise any type ofmolecule, such as, for example, protein, oligonucleotide,polysaccharide, or small molecule. In some embodiments, an antigencomprises more than one molecule, such as for example, a heterodimericreceptor, a receptor bound to its ligand, or a complex comprising aprotein and a small molecule or oligonucleotide. In some embodiments, atarget is an antigen.

The term “aqueous solution,” as used herein, refers to a solution thatis at least 50% water. In some embodiments, an aqueous solution retainsthe solution characteristics of water.

The term “azide reactive,” as used herein, refers to a chemical moietythat selectively reacts with an azide on another molecule to form acovalent chemical bond between the azido modified group and the azidereactive group. Examples of azide-reactive groups include, but are notlimited to, alkyne, including, but not limited to, terminal alkynes andactivated alkynes; and phosphines, including, but not limited to,triarylphosphines. “Azide-reactive” can also refer to a molecule thatcontains a chemical moiety that selectively reacts with an azido group.

The term “biomolecule,” as used herein, refers to proteins, peptides,amino acids, glycoproteins, nucleic acids, nucleotides, nucleosides,oligonucleotides, sugars, oligosaccharides, lipids, hormones,proteoglycans, carbohydrates, polypeptides, polynucleotides,polysaccharides, drugs, prodrugs, etc., which may be found in a livingorganism (including an isolated cell). A biomolecule need not be anaturally-occurring molecule, but may be a molecule that has beenintroduced into the living organism or an ancestor of the livingorganism, e.g., directly, through transgenic methods, or otherwise.

The term “carrier molecule,” as used herein, refers to a biological or anon-biological moiety that is covalently bonded to a compound of thepresent invention, and which confers a desirable property on thecompound and/or on a biomolecule conjugated thereto. Nonlimitingexemplary such desirable properties include binding properties, such as,for example, the ability to specifically bind to another moiety (e.g., amember of a binding pair); increasing half-life; increasing solubility;and directing the compound to a particular location in a cell ororganism. Such moieties include, but are not limited to, amino acids,peptides, proteins, polysaccharides, nucleosides, nucleotides,oligonucleotides, nucleic acids, haptens, psoralens, drugs, hormones,lipids, lipid assemblies, synthetic polymers, polymeric microparticles,biological cells, viruses, and combinations thereof.

The term, “chemical handle,” as used herein, refers to a functionalgroup that is capable of undergoing a click reaction, a 1,3-dipolarcycloaddition, and/or a Staudinger ligation. Nonlimiting exemplarychemical handles include alkyne-reactive moieties, such as azide; andazide-reactive moieties, such as alkynes, including, but not limited to,terminal alkynes and activated alkynes; and phosphines, including, butnot limited to, a triarylphosphine; and the like.

The term “complementary chemical handle,” as used herein, refers to afunctional group that is capable of undergoing a click reaction, a1,3-dipolar cycloaddition, and/or a Staudinger ligation with a specifiedchemical handle. For example, for an azide chemical handle,complementary chemical handles include, but are not limited to, alkynes,such as terminal alkynes and activated alkynes, and phosphines, such astriarylphosphines.

The terms “click chemistry” and “click reaction,” as used herein, referto copper ion-catalyzed 1,3-dipolar cycloadditions between an azide anda terminal alkyne to form a 1,2,3-triazole.

The term “1,3-dipolar cycloaddition,” as used herein, refers toreactions between an azide and an alkyne to form a 1,2,3-triazole.

The term “copper ion source,” as used herein, refers to any source ofCu(I) ions, whether or not formation of Cu(I) ions involves otheragents, such as reducing agents. Nonlimiting exemplary copper ionsources include copper salts, such as Cu(NO₃)₂ Cu(OAc)₂ or CuSO₄; copperhalides, such as CuBr and CuI; and copper-containing metals, such ascopper wire.

The terms “copper ion chelator” and “copper chelator,” as used herein,refer to a moiety that binds to, and stabilizes, Cu(I) ions. Nonlimitingexemplary copper chelators are discussed herein.

The term “halogen,” as used herein, refers to an atom selected from F,Cl, Br, and I.

The term “linker” or “L”, as used herein, refers to a single covalentbond or a series of stable covalent bonds incorporating 1-30 nonhydrogenatoms selected from the group consisting of C, N, O, S, P, Si, and Se.Exemplary linking members include moieties that includes —C(O)NH—,—C(O)O—, —NH—, —S—, —O—, and the like. In some embodiments, a linker hasa chain length of 1-30 atoms, or 1-25 atoms, or 1-20 atoms, or 1-15atoms, or 1-10 atoms, or 1-5 atoms. A “cleavable linker” is a linkerthat has one or more covalent bonds that can be broken under particularreaction conditions or in the presence of a particular molecule orenzyme, such that the moiety on one side of the cleavable linker is nolonger covalently bound to the moiety on the other side of the cleavablelinker. The term “cleavable group” refers to a moiety that allows forrelease of a portion, e.g., a reporter molecule, carrier molecule orsolid support, of a conjugate from the remainder of the conjugate bycleaving a bond linking the released moiety to the remainder of theconjugate. Such cleavage (for both cleavable linkers and cleavablegroups) is either chemical in nature, or enzymatically mediated.Exemplary enzymatically cleavable linkers and groups include naturalamino acids or peptide sequences that end with a natural amino acid. Inaddition to enzymatically cleavable linkers and groups, it is within thescope of the present invention to include one or more sites that arecleaved by the action of an agent other than an enzyme. Exemplarynon-enzymatic cleavage agents include, but are not limited to, acids,bases, light (e.g., nitrobenzyl derivatives, phenacyl groups, benzoinesters), and heat. Many cleavable groups are known in the art. See, forexample, Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshiet al., J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J.Immunol., 124: 913-920 (1980); Bouizar et al., Eur. J. Biochem., 155:141-147 (1986); Park et al., J. Biol. Chem., 261: 205-210 (1986);Browning et al., J. Immunol., 143: 1859-1867 (1989). Moreover a broadrange of cleavable, bifunctional (both homo- and hetero-bifunctional)spacer arms are commercially available. An exemplary cleavable linker orgroup, an ester, may be cleaved by a reagent, e.g. sodium hydroxide,resulting in a carboxylate-containing product and a hydroxyl-containingproduct.

The term “low copper,” as used herein, refers to a copper concentrationof less than 1 millimolar.

The term “modified biomolecule” as used herein refers to a biomoleculewhich has been modified by covalent attachment of at least one chemicalhandle. A biomolecule may be modified in vitro or in vivo.

The term “phosphine reactive” as used herein refers to a chemical moietythat selectively reacts via Staudinger ligation with a phosphine group,including but not limited to a triarylphosphine group, on anothermolecule to form a covalent chemical bond. Examples of phosphinereactive groups include, but are not limited to, azide.

The terms “protein” and “polypeptide” are used herein in a generic senseto refer to polymers of amino acid residues of any length. The term“peptide” is used herein to refer to polypeptides having fewer than 100amino acid residues, typically fewer than 10 amino acid residues. Theamino acid residues in a polypeptide, protein, or peptide may benaturally-occurring amino acid residues or non-naturally occurring aminoacid residues.

The term “reducing agent,” as used herein, refers to an agent that iscapable of reducing Cu(II) to Cu(I). Nonlimiting exemplary reducingagents include ascorbate, tris(2-carboxyethyl) phosphine (TCEP), NADH,NADPH, thiosulfate, metallic copper, hydroquinone, vitamin K₁,glutathione, cysteine, 2-mercaptoethanol, dithiothreitol, and an appliedelectric potential. Nonlimiting exemplary metals that may act asreducing agents include Al, Be, Co, Cr, Fe, Mg, Mn, Ni, Zn, Au, Ag, Hg,Cd, Zr, Ru, Fe, Co, Pt, Pd, Ni, Rh, and W.

The term “reporter molecule” refers to a moiety that is directly orindirectly detectable. In some embodiments, and as a non-limitingexample, a reporter molecule may be directly detectable, e.g., due toits spectral properties. In some embodiments, and as a non-limitingexample, a reporter molecule may be indirectly detectable, e.g., due toits enzymatic activity, wherein the enzymatic activity produces adirectly detectable signal. Such reporter molecules include, but are notlimited to, radiolabels; pigments, dyes, and other chromogens; spinlabels; fluorescent labels (i.e., fluorophores such as coumarins,cyanines, benzofurans, quinolines, quinazolinones, indoles, benzazoles,borapolyazaindacenes, and xanthenes, including fluoresceins, rhodamines,and rhodols); chemiluminescent substances, wherein the detectable signalis generated by chemical modification of substance; metal-containingsubstances; enzymes, wherein the enzyme activity generates a signal(such as, for example, by forming a detectable product from a substrate;haptens that can bind selectively to another molecule (such as, forexample, an antigen that binds to an antibody; or biotin, which binds toavidin and streptavidin). Many reporter molecules are known in the art,some of which are described, e.g., in Richard P. Haugland, MolecularProbes Handbook of Fluorescent Probes and Research Products (9^(th)edition, CD-ROM, September 2002), supra.

The term “solid support,” as used herein, refers to a material that issubstantially insoluble in a selected solvent system, or which can bereadily separated (e.g., by precipitation) from a selected solventsystem in which it is soluble. Solid supports useful in practicing thepresent invention may include groups that are activated or capable ofactivation such that one or more compounds described herein will bind tothe solid support.

The terms “structural integrity of the [biomolecule] is not reduced” or“preservation of the structural integrity of the [biomolecule]”, as usedherein, mean that either: 1) when analyzed by gel electrophoresis anddetection (such as staining), a band or spot arising from the labeledbiomolecule is not reduced in intensity by more than 20%, and preferablynot reduced by more than 10%, with respect to the corresponding band orspot arising from the same amount of the electrophoresed unlabeledbiomolecule, arising from the labeled biomolecule analyzed; or 2) whenanalyzed by gel electrophoresis, a band or spot arising from the labeledbiomolecule is not observed to be significantly less sharp than thecorresponding band or spot arising from the same amount of theelectrophoresed unlabeled biomolecule, where “significantly less sharp”(synonymous with “significantly more diffuse”) means the detectable bandor spot takes up at least 5% more, preferably 10% more, more preferably20% more area on the gel than the corresponding unlabeled biomolecule.Other reproducible tests for structural integrity of labeledbiomolecules include, without limitation detection of released aminoacids or peptides, or mass spectrometry.

The term “therapeutic molecule” refers to a molecule that can be used totreat and/or alleviate a condition and/or symptom in a subject, and/orcan be used to affect biological processes in cells in vitro.Therapeutic molecules include, but are not limited to, antimetabolites,alkylating agents, anthracyclines, antibiotics, and anti-mitotic agents.Nonlimiting exemplary therapeutic molecules include taxol, cytochalasinB, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine,tetracaine, lidocaine, propranolol, puromycin, abrin, ricin A,pseudomonas exotoxin, diphtheria toxin, tumor necrosis factor,γ-interferon, α-interferon, nerve growth factor, platelet derived growthfactor, tissue plasminogen activator, interleukin-1, interleukin-2,interleukin-6, granulocyte macrophage colony stimulating factor, orgranulocyte colony stimulating factor, and analogs or homologs thereof.

The present invention provides low-copper click reactions, 1,3-dipolarcycloadditions, and Staudinger ligations involving a modifiedbiomolecule and a compound of any one of Formulas (I) to (XIII). In someembodiments, the modified biomolecule comprises an azide moiety and thecompound of any one of Formulas (I) to (XIII) comprises a terminalalkyne. In some embodiments, the modified biomolecule comprises analkyne, such as a terminal alkyne or an activated alkyne, or aphosphine, such as a triarylphosphine, and the compound of any one ofFormulas (I) to (XIII) comprises an azide moiety.

Accordingly, provided herein are compounds, compositions, methods, andkits for the labeling, detecting, isolating and/or analysis ofbiomolecules. In some embodiments, presented are novel compoundscomprising an azide moiety or an alkyne moiety. In some embodiments,methods are provided for covalently attaching the novel compounds tomodified biomolecules using a click reaction, a 1,3-dipolarcycloaddition, or a Staudinger ligation. In some such embodiments, themethod comprises labeling, detecting, isolating and/or analyzing thebiomolecule.

Click Chemistry

Azides and terminal alkynes can undergo Cu(I)-catalyzed Azide-AlkyneCycloaddition (CuAAC) at room temperature. Such Cu(I)-catalyzedazide-alkyne cycloadditions, sometimes referred to as click chemistry,typically results in formation of a 1,2,3-triazole. Various exemplaryclick reactions are known in the art, and are described, e.g., in U.S.Publication No. 2005/0222427.

Click reactions can be performed in a variety of aqueous solutions,including, but not limited to, water, and mixtures of water and variousmiscible or partially miscible organic solvents. Nonlimiting suchorganic solvents include alcohols, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tert-butanol (tBuOH) and acetone.

In some embodiments, the copper used as a catalyst in a click reactionis Cu(I) ions. Exemplary sources of Cu(I) ions include, but are notlimited to, cuprous halides such as cuprous bromide or cuprous iodide.In some embodiments, a click reaction is carried out in the presence ofCu(II) ions and a reducing agent, which reduces the Cu(II) to Cu(I) insitu. Exemplary sources of Cu(II) ions include, but are not limited to,Cu(NO₃)₂, Cu(OAc)₂, and CuSO₄. Nonlimiting exemplary reducing agentsinclude ascorbate, tris(2-carboxyethyl) phosphine (TCEP), NADH, NADPH,thiosulfate, metallic copper, hydroquinone, vitamin K₁, glutathione,cysteine, 2-mercaptoethanol, dithiothreitol, Fe²⁺, Co²⁺, and an appliedelectric potential. In some embodiments, a reducing agent is a metalselected from Al, Be, Co, Cr, Fe, Mg, Mn, Ni, Zn, Au, Ag, Hg, Cd, Zr,Ru, Fe, Co, Pt, Pd, Ni, Rh, and W.

In some embodiments, the reducing agent is included in a click reactionin a micromolar to millimolar range. In some embodiments, theconcentration of the reducing agent is between 100 μM and 100 mM,between 10 μM and 10 mM, or between 1 μM and 1 mM.

In some embodiments, a click reaction includes a chelator thatstabilizes Cu(I) ions. Nonlimiting exemplary such chelators aredescribed herein.

In some embodiments, at least one copper chelator is included in a clickreaction. In some such embodiments, the copper chelator is added after aCu(II) source has been contacted with a reducing agent. In someembodiments, the copper chelator is added at the same time the Cu(II)source is contacted with a reducing agent. In some embodiments, a copperchelator is added to a solution containing one or both of the clickreactants (i.e., a solution containing one or both of theazide-containing reactant and the alkyne-containing reactant), and asolution containing the Cu(II) source and the reducing agent issubsequently added to initiate the click reaction.

In some embodiments, a click reaction comprises a compound of any one ofFormulas (I) to (XIII) and a modified biomolecule. In some suchembodiments, the compound of any one of Formulas (I) to (XIII) comprisesa terminal alkyne and the modified biomolecule comprises an azide. Insome embodiments, the compound of any one of formulas (I) to (XIII)comprises an azide and the modified biomolecule comprises a terminalalkyne. In some embodiments, a click reaction further comprises Cu(I)ions. In some embodiments, a click reaction further comprises Cu(II)ions and at least one reducing agent. In some embodiments, a clickreaction further comprises a copper chelator.

Activated Alkyne Chemistry (1,3-Dipolar Cycloadditions)

In some instances, azides and alkynes can undergo catalyst-free1,3-dipolar cycloaddition when an activated alkyne is used. In someembodiments, alkynes can be activated by ring strain such as, by way ofexample only, eight membered ring structures, including seven toten-membered ring structures with electron-withdrawing groups appendedthereon. In some embodiments, alkynes can be activated by the additionof a Lewis acid such as, by way of example only, Au(I) or Au(III).Nonlimiting exemplary activated alkynes include cyclooctynes anddifluorocyclooctynes, which are described, e.g., in Agard et al., J. Am.Chem. Soc., 2004, 126 (46):15046-15047; dibenzocyclooctynes, which aredescribed, e.g., in Boon et al., WO2009/067663 A1; andaza-dibenzocyclooctynes, which are described, e.g., in Debets et al.,Chem. Comm., 2010, 46:97-99.

Typically, an activated alkyne conjugated with fluorophores or antibodyundergoes cycloaddition to azide in one to twelve hour at roomtemperature. The reaction can be carried out in organic or aqueoussolvents, buffers like PBS, TRIS or mixtures of buffers and organicsolvents.

In some embodiments of the methods described herein, a modifiedbiomolecule comprises an activated alkyne and a compound of any one ofFormulas (I) to (XIII) comprises an azide.

Staudinger Ligation

In a Staudinger ligation, an azide is reacted with a triarylphosphinecomprising an electrophilic trap (typically, a methyl ester). Followingformation of an aza-ylide intermediate, the intermediate rearranges toproduce a ligated product having an amide linkage, and a phosphineoxide. Such ligations are described, e.g., in U.S. Publication No.2006/0276658. In some embodiments, the phosphine comprises an acyl groupsuch as an ester, thioester or N-acyl imidazole (i.e. a phosphinoester,phosphinothioester, phosphinoimidazole) to trap the aza-ylideintermediate and form an amide bond upon hydrolysis. In someembodiments, the phosphine can be a di- or triarylphosphine to stabilizethe phosphine. The phosphines used in Staudinger ligation methodsdescribed herein include, but are not limited to, cyclic or acyclic,halogenated, bisphosphorus, or polymeric phosphines.

A typical procedure for a Staudinger ligation is as follows (J. Am.Chem. Soc. 2002, 124, 14893-14902): The cells were pelleted (3500 rpm, 3min) and washed twice with 200 μL of labeling buffer (1% FBS in PBS, pH)7.4). After the second wash, cells were typically resuspended in avolume of 50 μL of labeling buffer and 50 μL of 2 in solution (0.5 mM inPBS, pH) 7.4). After incubation at room temperature for 1 h, the cellswere pelleted (3500 rpm, 3 min) and washed three times with ice-coldlabeling buffer, the cells were pelleted, washed with 200 μL of ice-coldlabeling buffer, and then diluted to a volume of 400 μL for flowcytometry analysis.

In some embodiments of the methods described herein, a modifiedbiomolecule comprises a phosphine and a compound of any one of Formulas(I) to (XIII) comprises an azide.

Compounds for Conjugating Biomolecules

In some embodiments, the present invention provides compounds having theformula:

wherein:

A is a carbon, or A, R₅, and R₆ are absent;

R₁, R₂, R₃, and R₄, are independently selected from hydrogen, halogen,—SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl, arylcarboxamido, alkyl and aryl portions areoptionally substituted one or more times by halogen, —SO₃X, a carboxylicacid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido; ortwo substituents selected from R₁, R₂, R₃, and R₄, wherein each of theat least two substituents are on different carbon atoms together form afused moiety selected from cycloalkyl, heterocycloalkyl, substitutedcycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substitutedaryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, andall of the remaining substituents are independently selected fromhydrogen, halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl; or at least two of the remaining substituentstogether form a fused moiety selected from cycloalkyl, heterocycloalkyl,substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl, and any remaining substituents are independently selectedfrom hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl;

R₅, and R₆, are independently selected from hydrogen, halogen, —SO₃X, acarboxylic acid, a salt of carboxylic acid, CN, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,alkoxy, substituted alkoxy, alkylthio, alkanoylamino,alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, arylcarboxamido,alkyl and aryl portions are optionally substituted one or more times byhalogen, —SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, nitro,hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl,heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, andsubstituted heteroaryl, arylcarboxamido;

at least one substituent selected from R₁, R₂, R₃, R₄, R₅ and R₆comprises X-L-, wherein:

-   -   X is selected from a reporter molecule, a carrier molecule, a        solid phase, a therapeutic molecule such as peptide, a protein,        an antibody, a polysaccharide, a nucleic acid polymer, an ion        complexing moiety, a lipid or a non-biological organic polymer        or polymeric micro or nano particle, that are optionally bound        to one or more additional fluorophores; or    -   X is a reactive group such as carboxylic acid, an activated        ester of carboxylic acid, an amine, a hydrazine, a        haloacetamide, an alkyl halide, an isothiocynate or a maleimide        group; and    -   L is an independently a single covalent bond or L is covalent        linkage having 1-24 non-hydrogen atoms selected from the group        consisting of C, N, O, P and S and composed of any combinations        of single, double, triple or aromatic carbon-carbon bonds,        carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen        bonds, carbon-sulfur bonds, phosphorus-oxygen bonds and        phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,        cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,        substituted alkyl, substituted heteroalkyl, substituted        cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,        substituted aryl, arylalkyl, substituted arylalkyl, and        substituted heteroaryl;

Z is an independently a single covalent bond or Z is covalent linkagehaving 1-10 non-hydrogen atoms selected from the group consisting of C,N, O, P and S and composed of any combinations of single, double, tripleor aromatic carbon-carbon bonds, carbon-nitrogen bonds, carbon-oxygenbonds, and carbon-sulfur bonds in the form of a straight- orbranched-chain alkyl or heteroalkyl chain;

G is a chemical handle selected from an azide-reactive group, analkyne-reactive group, and a phosphine-reactive group.

In some embodiments, the compound is of the formula:

In some embodiments, the compound is of the formula:

wherein

R₂, R₃, R₄, and R₇ to R₁₂ are independently selected from hydrogen,halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substitutedalkyl, substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl; or two substituents selected from R₂, R₃, R₄, and R₇ to R₁₂,wherein the two substituents are on different carbon atoms, togetherform a fused moiety selected from cycloalkyl, heterocycloalkyl,substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl, and all of the remaining substituents are independentlyselected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl; or two of theremaining substituents also together form a fused moiety selected fromcycloalkyl, heterocycloalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, and the remainingsubstituents are independently selected from hydrogen, halogen, alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl.

In some embodiments, the present invention provides compounds having theformula:

wherein:

R₁, R₂, R₃, and R₄ are independently selected from hydrogen, halogen,alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl; or two substituents selected from R₁, R₂, R₃, and R₄together form a fused moiety selected from cycloalkyl, heterocycloalkyl,substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl, and the remaining two substituents also together form afused moiety selected from cycloalkyl, heterocycloalkyl, substitutedcycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substitutedaryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, orthe remaining two substituents are independently selected from hydrogen,halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substitutedalkyl, substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl;

at least one substituent selected from R₁, R₂, R₃, R₄, R₅, and R₆comprises X-L-, wherein:

-   -   X is selected from a reporter molecule, a carrier molecule, a        solid phase, or a therapeutic molecule; and    -   L is a group selected from alkyl, heteroalkyl, cycloalkyl,        heterocycloalkyl, alkoxy, substituted alkoxy, substituted alkyl,        substituted heteroalkyl, substituted cycloalkyl, substituted        heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,        substituted arylalkyl, and substituted heteroaryl, having a        chain length of 0-20 atoms;    -   A is a carbon, and R₅ and R₆ are independently selected from        hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,        heterocycloalkyl, substituted alkyl, substituted heteroalkyl,        substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,        substituted alkoxy, aryl, heteroaryl, substituted aryl,        arylalkyl, substituted arylalkyl, and substituted heteroaryl; or        A, R5, and R6 are absent;

B is selected from O, S, and NR₇, wherein R₇ is selected from hydrogen,halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substitutedalkyl, substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl

Z is a straight- or branched-chain alkyl or heteroalkyl having a chainlength of 1-10 atoms, or is absent; and

G is a chemical handle selected from an azide-reactive group, analkyne-reactive group, and a phosphine-reactive group.

In some embodiments, the present invention provides compounds having theformula:

wherein:

A is a carbon, or A, R₅, and R₆ are absent;

R₁, R₂, R₅, and R₆ are independently selected from hydrogen, halogen,alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl; or two substituents selected from R₁, R₂, R₅, and R₆,wherein the two substituents are on different carbon atoms, togetherform a fused moiety selected from cycloalkyl, heterocycloalkyl,substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl, and the remaining substituents are independently selectedfrom hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl; at least onesubstituent selected from R₁, R₂, R₅, and R₆ comprises X-L-, wherein:

-   -   X is selected from a reporter molecule, a carrier molecule, a        solid phase, or a therapeutic molecule; and    -   L is a group selected from alkyl, heteroalkyl, cycloalkyl,        heterocycloalkyl, alkoxy, substituted alkoxy, substituted alkyl,        substituted heteroalkyl, substituted cycloalkyl, substituted        heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,        substituted arylalkyl, and substituted heteroaryl, having a        chain length of 0-20 atoms;

B is selected from O, S, and NR₃, wherein R₃ is selected from hydrogen,halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substitutedalkyl, substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl;

Z is a straight- or branched-chain alkyl or heteroalkyl having a chainlength of 1-10 atoms, or is absent; and

G is a chemical handle selected from an azide-reactive group, analkyne-reactive group, and a phosphine-reactive group.

In some embodiments, when a compound is of Formula (VI), R₁, R₂, R₅, andR₆ are independently selected from hydrogen, halogen, alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl; or R₁ and R₂ together form a fused moiety selected fromcycloalkyl, heterocycloalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl, and R₅ and R₆ areindependently selected from hydrogen, halogen, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,alkoxy, substituted alkoxy, aryl, heteroaryl, substituted aryl,arylalkyl, substituted arylalkyl, and substituted heteroaryl.

In some embodiments, the present invention provides compounds having theformula:

wherein:

A is a carbon, or A, R₅, and R₆ are absent;

R₁, R₂, R₃, R₅, and R₆ are independently selected from hydrogen,halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substitutedalkyl, substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl; or two substituents selected from R₁, R₂, R₃, R₅, and R₆,wherein the two substituents are on different carbon atoms, togetherform a fused moiety selected from cycloalkyl, heterocycloalkyl,substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl, and the remaining substituents are selected from hydrogen,halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substitutedalkyl, substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl;

at least one substituent selected from R₁, R₂, R₃, R₅, and R₆ comprisesX-L-, wherein:

-   -   X is selected from a reporter molecule, a carrier molecule, a        solid phase, or a therapeutic molecule; and    -   L is a group selected from alkyl, heteroalkyl, cycloalkyl,        heterocycloalkyl, alkoxy, substituted alkoxy, substituted alkyl,        substituted heteroalkyl, substituted cycloalkyl, substituted        heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,        substituted arylalkyl, and substituted heteroaryl, having a        chain length of 0-20 atoms;

Z is a straight- or branched-chain alkyl or heteroalkyl having a chainlength of 1-10 atoms, or is absent; and

G is a chemical handle selected from an azide-reactive group, analkyne-reactive group, and a phosphine-reactive group.

In some embodiments, the present invention provides compounds having theformula:

wherein:

A is a carbon, or A, R₅, and R₆ are absent;

R₁, R₂, R₅, and R₆ are independently selected from hydrogen, halogen,alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl; at least one substituent selected from R₁, R₂, R₅, and R₆comprises X-L-, wherein:

-   -   X is selected from a reporter molecule, a carrier molecule, a        solid phase, or a therapeutic molecule; and    -   L is a group selected from alkyl, heteroalkyl, cycloalkyl,        heterocycloalkyl, alkoxy, substituted alkoxy, substituted alkyl,        substituted heteroalkyl, substituted cycloalkyl, substituted        heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,        substituted arylalkyl, and substituted heteroaryl, having a        chain length of 0-20 atoms;        Z is a straight- or branched-chain alkyl or heteroalkyl having a        chain length of 1-10 atoms, or is absent; and

G is a chemical handle selected from an azide-reactive group, analkyne-reactive group, and a phosphine-reactive group.

In some embodiments, the present invention provides compounds having theformula:

wherein:

A is a carbon, or A, R₅, and R₆ are absent;

m and n is an integer between 4 and 8;

R₇ is selected from hydrogen, alkyl, heteroalkyl, substituted alkyl, andsubstituted heteroalkyl;

R₁, R₂, R₃, R₄, R₅, R₆, each R′, and each R″ are independently selectedfrom hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,substituted arylalkyl, and substituted heteroaryl; or two substituentsselected from R₁, R₂, R₃, R₄, R₅, R₆, an R′, and an R″, wherein the twosubstituents are on different carbon atoms, together form a fused moietyselected from cycloalkyl, heterocycloalkyl, substituted cycloalkyl,substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,arylalkyl, substituted arylalkyl, and substituted heteroaryl, and all ofthe remaining substituents are independently selected from hydrogen,halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substitutedalkyl, substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl; or two of the remaining substituents also together form afused moiety selected from cycloalkyl, heterocycloalkyl, substitutedcycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substitutedaryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, andthe remaining substituents are independently selected from hydrogen,halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substitutedalkyl, substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,substituted aryl, arylalkyl, substituted arylalkyl, and substitutedheteroaryl;

at least one substituent selected from R₁, R₂, R₃, R₄, R₅, R₆, an R′,and an R″ comprises X-L-, wherein:

-   -   X is selected from a reporter molecule, a carrier molecule, a        solid phase, or a therapeutic molecule; and    -   L is a group selected from alkyl, heteroalkyl, cycloalkyl,        heterocycloalkyl, alkoxy, substituted alkoxy, substituted alkyl,        substituted heteroalkyl, substituted cycloalkyl, substituted        heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,        substituted arylalkyl, and substituted heteroaryl, having a        chain length of 0-20 atoms;

Z is a straight- or branched-chain alkyl or heteroalkyl having a chainlength of 1-10 atoms, or is absent; and

G is a chemical handle selected from an azide-reactive group, analkyne-reactive group, and a phosphine-reactive group.

In some embodiments of compounds of Formulas (I) to (XIII), one of the Rsubstituents comprises X-L-, and the remaining R substituents are eachH. In some embodiments compounds of Formulas (I) to (XIII), L is analkyl group having a chain length of 0 to 15 atoms, 0 to 10 atoms, or 0to 5 atoms. In some embodiments compounds of Formulas (I) to (XIII), Lis —NH—(CH₂)_(n)—NH—C(O)—, wherein n is 1 to 12. In some embodiments, nis 1 to 10, 1 to 8, or 1 to 5.

In some embodiments compounds of Formulas (I) to (XIII), G is an azideor a terminal alkyne. In some embodiments, when a compound of any one ofFormulas (I) to (XIII) is to be used in a click reaction, G is an azideor terminal alkyne. In some embodiments, when a compound of any one ofFormulas (I) to (XIII) is to be used in a 1,3-dipolar cycloaddition withan activated alkyne, G is an azide. In some embodiments, when a compoundof any one of Formulas (I) to (XIII) is to be used in a Staudingerligation, G is an azide.

In some embodiments, the reporter molecule comprises a chromophore,fluorophore, fluorescent protein, phosphorescent dye, tandem dye,particle, hapten, enzyme, or radioisotope. In some embodiments, thefluorophore is a xanthene, coumarin, cyanine, pyrene, oxazine,borapolyazaindacene, or carbopyranine. In some embodiments, the enzymeis horseradish peroxidase, alkaline phosphatase, beta-galactosidase, orbeta-lactamase. In some embodiments, the particle is a semiconductornanocrystal.

In some embodiments, the carrier molecule is an amino acid, peptide,protein, polysaccharide, nucleoside, nucleotide, oligonucleotide,nucleic acid, hapten, psoralen, drug, hormone, lipid, lipid assembly,tyramine, synthetic polymer, polymeric microparticle, biological cell,cellular component, ion chelating moiety, enzymatic substrate, or virus.In some embodiments, the carrier molecule is an antibody, antibodyfragment, antigen, avidin, streptavidin, biotin, dextran, IgG bindingprotein, fluorescent protein, agarose, or non-biological microparticle.

In some embodiments, the solid support is an aerogel, hydrogel, resin,bead, biochip, microfluidic chip, silicon chip, multi-well plate,membrane, conducting metal, nonconducting metal, glass, or magneticsupport. In some embodiments, the solid support is a silica gel,polymeric membrane, particle, derivatized plastic film, glass bead,cotton, plastic bead, alumina gel, polysaccharide, poly(acrylate),polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose,dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin,nitrocellulose, diazocellulose, polyvinylchloride, polypropylene,polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead,superparamagnetic bead, or starch.

In some embodiments, the therapeutic molecule is taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine,tetracaine, lidocaine, propranolol, puromycin, or analogs or homologsthereof. In some embodiments, the therapeutic molecule is anantimetabolite, alkylating agent, anthracycline, antibiotic, oranti-mitotic agent. In some embodiments, the therapeutic molecule isabrin, ricin A, pseudomonas exotoxin, diphtheria toxin, tumor necrosisfactor, γ-interferon, α-interferon, nerve growth factor, plateletderived growth factor, tissue plasminogen activator, interleukin-1,interleukin-2, interleukin-6, granulocyte macrophage colony stimulatingfactor, or granulocyte colony stimulating factor.

The compounds of the present invention may be made, for example, usingthe exemplary reaction schemes shown in FIG. 1 and FIG. 2, and describedin Example 1.

Reporter Molecules

In some embodiments, a compound of any one of Formulas (I) to (XIII)comprises a reporter molecule. The reporter molecules used in themethods and compositions provided herein include any directly orindirectly detectable reporter molecule that can be covalently attachedas a substituent of a compound of any one of Formulas (I) to (XIII).

Reporter molecules used in the methods and compositions described hereininclude, but are not limited to, chromophores, fluorophores, fluorescentproteins, phosphorescent dyes, tandem dyes, particles, haptens, enzymes,and radioisotopes. In some embodiments, a reporter molecule is afluorophore, a fluorescent protein, a hapten, or an enzyme.

A fluorophore is any chemical moiety that exhibits an absorption maximumat wavelengths greater than 280 nm, and retains its spectral propertieswhen covalently attached to a biomolecule following reaction of acompound of any one of Formulas (I) to (XIII) comprising the fluorophorewith the modified biomolecule. Fluorophores include, without limitation,pyrenes; anthracenes; naphthalenes; acridines; stilbenes; indoles andbenzindoles; oxazoles and benzoxazoles; thiazoles and benzothiazoles;4-amino-7-nitrobenz-2-oxa-1,3-diazoles (NBD); cyanines; carbocyanines;carbostyryls; porphyrina; salicylates; anthranilates; azulenes;perylenes; pyridines; quinolines; borapolyazaindacenes; xanthenes(including, but not limited to, fluoresceins (such as benzo- ordibenzofluoresceins, seminaphthofluoresceins, or naphthofluoresceins),rhodols (such as eminaphthorhodafluors), and rhodamine); oxazines andbenzoxazines (including, but not limited to, resorufins,aminooxazinones, diaminooxazines, and their benzo-substituted analogs);carbazines; phenalenones; coumarins; benzofurans; benzphenalenones;carbopyranines, semiconductor nanocrystals; and derivatives of any ofthe above.

In some embodiments, a reporter molecule is selected from a xanthene(including, but not limited to, sulfonated xanthenes, fluorinatedxanthenes, rhodol, rhodamine, fluorescein and derivatives thereof),coumarin (including, but not limited to, sulfonated coumarin andfluorinated coumarin), cyanine (including, but not limited to,sulfonated cyanine), pyrene, oxazine, borapolyazaindacene,carbopyranine, and semiconductor nanocrystal.

In some embodiments, a reporter molecule is a xanthene that is bound asa substituent of a compound of any one of Formulas (I) to (XIII) via asingle covalent bond at the 9-position of the xanthene. In someembodiments, the xanthene is selected from 3H-xanthen-6-ol-3-oneattached through the 9-position, 6-amino-3H-xanthen-3-one attachedthrough the 9-position, and 6-amino-3H-xanthen-3-imine attached throughthe 9-position.

One skilled in the art can select a fluorophore to be included as asubstituent of a compound of any one of Formulas (I) to (XIII) accordingto the particular application. Physical properties of a fluorophore thatcan be used for detection of modified biomolecules include, but are notlimited to, spectral characteristics (absorption, emission, and stokesshift), fluorescence intensity, lifetime, polarization andphoto-bleaching rate, and combinations thereof. In various embodiments,one or more of the physical properties can be used to distinguish onefluorophore from another, and thereby allow for multiplexed analysis. Insome embodiments, the fluorophore has an absorption maximum atwavelengths greater than 480 nm, at wavelengths between 488 nm to 514 nm(particularly suitable for excitation by the output of the argon-ionlaser excitation source), or at wavelengths near 546 nm (particularlysuitable for excitation by a mercury arc lamp).

Many of fluorophores can also function as chromophores and thus thedescribed fluorophores may also be used as chromophore reportermolecules in the methods and compositions described herein.

In some embodiments, a reporter molecule is an enzyme. In someembodiments, an enzyme is a desirable label because it can amplify thedetectable signal, thus increasing assay sensitivity. In someembodiments, the enzyme itself is not directly detectable, but itsactivity can be used to create a detectable signal when the enzyme iscontacted with an appropriate substrate, such that the convertedsubstrate produces, for example, a fluorescent, colorimetric, orluminescent signal. Various substrates are known in the art, some ofwhich are described in the Molecular Probes Handbook, supra.

In some embodiments, when an enzyme reporter molecule is anoxidoreductase such as, by way of example only, horseradish peroxidase,suitable substrates include, but are not limited to,3,3′-diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole (AEC),2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB),o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol,4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reducedbenzothiazines, including Amplex® Red reagent and its variants (U.S.Pat. No. 4,384,042), Amplex UltraRed and its variants (WO05/042504),reduced dihydroxanthenes, including dihydrofluoresceins anddihydrorhodamines, including dihydrorhodamine 123. Peroxidase substratesthat may be used with the enzymatic reporter molecules described hereinalso include, but are not limited to, tyramides (U.S. Pat. Nos.5,196,306; 5,583,001 and 5,731,158), which can be intrinsicallydetectable before action of the enzyme but are “fixed in place” by theaction of a peroxidase in the process described as tyramide signalamplification (TSA). In various embodiments, such substrates may beused, for example, to label targets in samples that are cells, tissuesor arrays for their subsequent detection by microscopy, flow cytometry,optical scanning and fluorometry.

In some embodiments, when an enzyme reporter molecule is a phosphataseenzyme such as, by way of example only, an acid phosphatases or analkaline phosphatase, suitable substrates include, but are not limitedto, 5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolylphosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenylphosphate, and o-nitrophenyl phosphate. Nonlimiting fluorogenicsubstrates include, but are not limited to, 4-methylumbelliferylphosphate, 6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP,U.S. Pat. No. 5,830,912), fluorescein diphosphate, 3-O-methylfluoresceinphosphate, resorufin phosphate,9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAOphosphate), ELF 97, ELF 39, and related phosphates (U.S. Pat. Nos.5,316,906 and 5,443,986).

In some embodiments, when an enzyme reporter molecule is a glycosidasesuch as, by way of example only, a beta-galactosidase,beta-glucuronidase, or beta-glucosidase, suitable substrates include,but are not limited to, 5-bromo-4-chloro-3-indolylbeta-D-galactopyranoside (X-gal) and similar indolyl galactosides,glucosides, and glucuronides, o-nitrophenyl beta-D-galactopyranoside(ONPG), p-nitrophenyl beta-D-galactopyranoside, resorufinbeta-D-galactopyranoside, fluorescein digalactoside (FDG), fluoresceindiglucuronide and their structural variants, 4-methylumbelliferylbeta-D-galactopyranoside, carboxyumbelliferyl beta-D-galactopyranoside,and fluorinated coumarin beta-D-galactopyranosides.

Enzyme reporter molecules also include, but are not limited to,hydrolases such as cholinesterases and peptidases, oxidases such asglucose oxidase and cytochrome oxidases, and reductases, for whichsuitable substrates are known. Additional nonlimiting exemplary enzymereporter molecules include luciferases and aequorins. In addition, thechemiluminescence-producing substrates for phosphatases, glycosidasesand oxidases such as those containing stable dioxetanes, luminol,isoluminol and acridinium esters can also be used with the enzymereporter molecules described herein.

In some embodiments, a reporter molecule is a hapten. Nonlimitingexemplary haptens include hormones, naturally occurring and syntheticdrugs, pollutants, allergens, affector molecules, growth factors,chemokines, cytokines, lymphokines, amino acids, peptides, chemicalintermediates, nucleotides, biotin and the like. In some embodiments, ahapten is not directly detectable, but it can bind to another moleculethat is detectable. As a nonlimiting example, a hapten may be an antigenthat can be bound by an antibody specific to that antigen, wherein theantibody comprises a detectable label, or wherein the antibody can bebound by a secondary antibody comprising a detectable label.

In some embodiments, a reporter molecule is a fluorescent protein.Nonlimiting exemplary fluorescent proteins include green fluorescentprotein (GFP) and the phycobiliproteins and derivatives thereof. In someembodiments, a fluorescent protein is used in conjunction with afluorophore in order to obtain a larger stokes shift from thefluorescent protein's absorption spectra. In some embodiments, thefluorescent protein and fluorophore function as an energy transfer pair,wherein the fluorescent protein emits at the wavelength at which thefluorophore absorbs and the fluorophore then emits at a wavelengthfarther from the fluorescent protein's emission wavelength than couldhave been obtained with only the fluorescent protein. In some suchembodiments, a compound of any one of Formulas (I) to (XIII) comprises afluorescent protein as one substituent and a fluorophore as anothersubstituent. In some embodiments, a compound of any one of Formulas (I)to (XIII) comprises both the fluorescent protein and the fluorophore asa single substituent, wherein the fluorescent protein and thefluorophore are connected to one another by a linker. Nonlimitingexemplary fluorescent protein/fluorophore pairs includephycobiliproteins and sulforhodamine fluorophores, sulfonated cyaninefluorophores, or sulfonated xanthene fluorophores. In some embodiments,the fluorophore functions as the energy donor and the fluorescentprotein as the energy acceptor. Nonlimiting exemplary radioisotopes thatmay be used as reporter molecules include For example, the compounds maybe radiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C), sulfur-35 (³⁵S), etc. Allisotopic variations of the compounds of the present invention, whetherradioactive or not, are intended to be encompassed within the scope ofthe present invention.

Methods of attaching reporter molecules as substituents of compounds ofFormulas (I) to (XIII) are known in the art. Nonlimiting exemplarymethods include the methods shown in FIGS. 1 and 2, in which a reportermolecule comprising an N-hydroxysuccinimidyl (NHS) ester is reacted witha precursor of a compound of any one of Formulas (I) to (XIII) bearing aprimary amine on at least one substituent. SDP esters, TFP, PFP,carbamates, thiocarbamates and maleimides may also be used in place ofNHS esters.

Carrier Molecules

In some embodiments, a compound of any one of Formulas (I) to (XIII)comprises a carrier molecule as a substituent.

Carrier molecules include, but are not limited to, antigens, steroids,vitamins, drugs, haptens, metabolites, toxins, environmental pollutants,amino acids, peptides, proteins, nucleic acids, nucleic acid polymers,carbohydrates, lipids, and polymers. In some embodiments, a carriermolecule comprises an amino acid, a peptide, a protein, an antibody orfragment thereof, an antigen, avidin, streptavidin, biotin, a dextran,an IgG binding protein (such as protein A or protein G), agarose, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipidassembly, a synthetic polymer, a non-biological microparticle (such as apolymeric microparticle), an ion chelating moiety, an enzymaticsubstrate, a biological cell, a cellular component, a virus, orcombinations thereof.

In some embodiments, when the carrier molecule is an enzymaticsubstrate, the enzymatic substrate is selected from an amino acid, apeptide, a sugar, an alcohol, alkanoic acid, 4-guanidinobenzoic acid, anucleic acid, a lipid, sulfate, phosphate, —CH₂OCO-alkyl, andcombinations thereof. In certain embodiments, such enzyme substrates canbe cleaved by enzymes selected from peptidases, phosphatases,glycosidases, dealkylases, esterases, guanidinobenzotases, sulfatases,lipases, peroxidases, histone deacetylases, exonucleases, reductases,endoglycoceramidases and endonucleases.

In some embodiments, when the carrier molecule comprises an amino acid,a peptide, or protein, the carrier molecule is selected from aneuropeptide, a cytokine, a toxin, a protease substrate, and a proteinkinase substrate. In some embodiments, a carrier is a peptide thatfunctions as an organelle localization peptide, that is, a peptide thatserves to target the conjugated compound for localization within aparticular cellular substructure by cellular transport mechanisms,including, but not limited to, a nuclear localization signal sequence.

In some embodiments, a carrier molecule is a protein selected from anenzyme, an antibody, a lectin, a glycoprotein, a histone, an albumin, alipoprotein, protein A, protein G, a phycobiliprotein or otherfluorescent protein, a hormone, a toxin, and a growth factor. In someembodiments, a carrier molecule is a protein selected from an antibody,an antibody fragment, avidin, streptavidin, a toxin, a lectin, or agrowth factor. In some embodiments, a carrier molecule comprises ahapten such as, for example, biotin, digoxigenin, or a fluorophore.

In some embodiments, a carrier molecule comprises a nucleic acid base,nucleoside, nucleotide or a nucleic acid polymer, a peptide nucleic acid(PNA), or a locked nucleic acid (LNA), single- or multi-stranded,natural or synthetic DNA or RNA oligonucleotide, or DNA/RNA hybrid,optionally containing an additional linker or spacer for attachment of afluorophore or other ligand. In some embodiments, a nucleic acid carriermolecule (including, but not limited to, LNA, PNA, DNA, and RNA)comprises fewer than 50 nucleotides, or fewer than 25 nucleotides.

In some embodiments, a carrier molecule comprises a carbohydrate orpolyol, including a polysaccharide, such as dextran, FICOLL, heparin,glycogen, amylopectin, mannan, inulin, starch, agarose and cellulose, ora polymer such as a poly(ethylene glycol). In some embodiments, acarrier molecule comprises dextran, agarose, or FICOLL.

In some embodiments, a carrier molecule comprises a lipid including, butnot limited to, glycolipids, phospholipids, and sphingolipids. In someembodiments, such lipids contain 6-25 carbons. In some embodiments, acarrier molecule includes a lipid vesicle, such as a liposome, or is alipoprotein. Some lipophilic substituents are useful, in someembodiments, for facilitating transport of a conjugated molecule intocells or cellular organelles.

In some embodiments, a carrier molecule is a cell, cellular fragment, orsubcellular particle, including virus particles, bacterial particles,virus components, biological cells (such as animal cells, plant cells,bacteria, or yeast), or cellular components. Non-limiting examples ofsuch cellular components include lysosomes, endosomes, cytoplasm,nuclei, histones, mitochondria, Golgi apparatus, endoplasmic reticulumand vacuoles.

In some embodiments, a carrier molecule comprises a specific bindingpair member. In some such embodiments, the presence of the carriermolecule, and therefore the biomolecule to which it is conjugatedthrough a compound of any one of Formulas (I) to (XIII), can be detectedusing a complementary specific binding pair member comprising adetectable label. Nonlimiting exemplary binding pairs are set forth inTable 2.

TABLE 2 Exemplary Specific Binding Pairs Antigen Antibody Biotin avidin(or streptavidin or anti-biotin) IgG* protein A or protein G Drug drugreceptor Folate folate binding protein Toxin toxin receptor Carbohydratelectin or carbohydrate receptor Peptide peptide receptor Protein proteinreceptor enzyme substrate Enzyme DNA (RNA) cDNA (cRNA)† Hormone hormonereceptor Ion Chelator *IgG is an immunoglobulin †cDNA and cRNA are thecomplementary strands used for hybridization

In some embodiments, a carrier molecule is an antibody-binding moiety,such as, but not limited to, anti-Fc, an anti-Fc isotype, anti-J chain,anti-kappa light chain, anti-lambda light chain, or a single-chainfragment variable protein, an anti-Fc Fab fragment; or a non-antibodypeptide or protein, such as, for example but not limited to, soluble Fcreceptor, protein G, protein A, protein L, lectins, or a fragmentthereof.

Methods of attaching carrier molecules as substituents of compounds ofFormulas (I) to (XIII) are known in the art. Nonlimiting exemplarymethods include as examples amides, thioamides, ethers, thioethers,carbamates, thiocarbamates, sulfhydryl groups, amino groups, etc.

Solid Supports

In some embodiments, a compound of any one of Formulas (I) to (XIII)comprises a solid support as a substituent.

A large number of solid supports are known in the art and can be used,in some embodiments, as a substituent of a compound of any one ofFormulas (I) to (XIII). Nonlimiting exemplary solid supports includesolid and semi-solid matrixes, such as aerogels and hydrogels, resins,beads, biochips (including thin film coated biochips), microfluidicchip, a silicon chip, multi-well plates (also referred to as microtiterplates or microplates), membranes, conducting and nonconducting metals,glass (including microscope slides) and magnetic supports. Othernonlimiting examples of solid supports include silica gels, polymericmembranes, particles, derivatized plastic films, derivatized glass,derivatized silica, glass beads, cotton, plastic beads, alumina gels,polysaccharides such as Sepharose, poly(acrylate), polystyrene,poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch,FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose,diazocellulose, polyvinylchloride, polypropylene, polyethylene(including poly(ethylene glycol)), nylon, latex bead, magnetic bead,paramagnetic bead, superparamagnetic bead, starch and the like. In someembodiments, the solid supports used in the methods and compositionsdescribed herein are substantially insoluble in liquid phases.

In some embodiments, a solid support may comprise a reactive functionalgroup, including, but not limited to, hydroxyl, carboxyl, amino, thiol,aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate,isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide, azide, alkyne,or phosphine, wherein such functional groups are used to covalentlyattach the solid support to a precursor of a compound of any one ofFormulas (I) to (XIII).

A suitable solid phase support used in the methods and compositionsdescribed herein, can be selected on the basis of desired use. By way ofexample only, where amide bond formation is desirable to attach theprecursor of a compound of any one of Formulas (I) to (XIII) to thesolid support, resins generally useful in peptide synthesis may beemployed, such as polystyrene, POLYHIPE™ resin, polyamide resin,polystyrene resin grafted with polyethylene glycol,polydimethyl-acrylamide resin, or PEGA beads. In some embodiments,precursors to compounds of Formulas (I) to (XIII) are deposited onto asolid support in an array format. In some such deposition isaccomplished by direct surface contact between the support surface and adelivery mechanism, such as a pin or a capillary, or by ink jettechnologies which utilize piezoelectric and other forms of propulsionto transfer liquids from miniature nozzles to solid surfaces.

Modified Biomolecules

The modification of biomolecules to incorporate chemical handles allowschemical attachment of another moiety (such as a reporter molecule orsolid support) through a subsequent click reaction. In some embodiments,the chemical handle of the modified biomolecule is selected from azide,alkyne (such as a terminal alkyne or an activated alkyne), andphosphine. In some embodiments, a biomolecule is modified in vivo, forexample, using cellular biosynthetic pathways, such as, for example,glycosylation of proteins, DNA replication, or transcription of RNA. Insome embodiments, a biomolecule is modified in vivo by contacting a cellwith a reagent that modifies a particular biomolecule or class ofbiomolecules. In some embodiments, a biomolecule is modified in vitrousing a reagent that modifies a biomolecule.

Various methods and reagents for modifying biomolecules in vivo areknown in the art. For example, in some embodiments, glycoproteins may bemodified in vivo by contacting a cell with non-native glycans thatcomprise chemical handles. The non-native glycans are used by the cellto glycosylate glycoproteins, resulting in covalent attachment ofchemical handles to such glycoproteins. Nonlimiting exemplary non-nativeglycans that may be used to modify glycoproteins with chemical handlesinclude tetraacetylated N-azidoacetylglucosamine, tetraacetylatedN-azidoacetylgalactosamine, tetraacetylated N-azidoacetylmannosamine,and tetraacetylfucose alkyne.

In some embodiments, a protein may be modified by incorporatingnon-native amino acids comprising chemical handles. Such modificationmay occur in vivo, during protein synthesis, or in an in vitro proteintranslation system. Nonlimiting exemplary non-native amino acids thatmay be used to modify proteins with chemical handles include, but arenot limited to, 4-azido-L-phenylalanine, L-azidohomoalanine, andL-homopropargylglycine.

In some embodiments, a prenylated protein may be modified, for example,by contacting a cell with a farnesyl alcohol azide or a geranylgeranylalcohol azide.

In some embodiments, a protein may be modified during fatty acidacylation of the protein, for example, by contacting a cell with anon-native fatty acid comprising a chemical handle. Nonlimitingexemplary non-native fatty acids that may be used to modify proteinswith chemical handles include, but are not limited to, palmitic acidazide, myristic acid azide, and the fatty acid analogs described, e.g.,in International Application No. PCT/US10/61768.

In some embodiments, DNA may be modified in vivo or in vitro usingvarious non-native nucleoside triphosphates that comprise chemicalhandles. In some embodiments, the DNA is modified during replicationthrough incorporation of a non-native nucleoside by DNA polymerase. Insome embodiments, the DNA is modified during apoptosis throughincorporation of a non-native nucleoside by terminal nucleotidyltransferase (TdT). Nonlimiting exemplary such non-native nucleosidetriphosphates include C-8-alkyne-dUTP and/or C8-alkyne-dCTP. Followingincorporation, the DNA comprises one or more covalently attached alkynegroups. In some embodiments, DNA may be modified during chemical DNAsynthesis using, for example, phophoramidites comprising chemicalhandles.

In some embodiments, RNA may be modified in vivo or in vitro usingvarious non-native nucleoside triphosphates that comprise chemicalhandles. In some embodiments, the RNA is modified during replicationthrough incorporation of a non-native nucleoside by RNA polymerase.Nonlimiting exemplary such non-native nucleoside triphosphates includeC-8-alkyne-UTP and/or C8-alkyne-CTP. Following incorporation, the RNAcomprises one or more covalently attached alkyne groups. In someembodiments, RNA may be modified during chemical RNA synthesis using,for example, phophoramidites comprising chemical handles.

In some embodiments, a biomolecule may be modified in vitro using areagent that covalently attaches a chemical handle through a particulargroup on the biomolecule. For example, in some embodiments, abiomolecule that comprises a primary amine (—NH₂) may be modified usinga reagent such as NHS-azide, NHS-phosphine, and sulfo-NHS-phosphine,SDP-azide, TFP-azide, PFP-azide, carbamate-azide, thiocarbamate-azideand maleimide-azide may also be used in place of NHS-azides.

Copper Ion Sources

In some embodiments, a click reaction comprises a copper ion source thatprovides Cu(I) ions. In some embodiments, a copper ion source providesCu(I) ions in the presence of a reducing agent. In some suchembodiments, a copper ion source provides Cu(II) ions, which are reducedto Cu(I) ions in the presence of a reducing agent. Nonlimiting exemplarycopper ion sources that produce Cu(I) ions include CuBr, CuI,tetrakis(acetonitrile)Cu(I) hexafluorophosphate,tetrakis(acetonitrile)Cu(I) tetrafluoroborate,tetrakis(acetonitrile)Cu(I) triflate, CuCN, Cu(I) butanethiolate, Cu(I)thiophenolate, Cu(I) triflate. In some embodiments, a copper ion sourcethat produces Cu(I) ions is included in a click reaction at aconcentration between 0.01 mM and 10 mM, between 0.01 mM and 5 mM,between 0.05 mM and 5 mM, between 0.1 mM and 5 mM, between 0.5 mM and 5mM, between 0.5 mM and 4 mM, or between 0.5 mM and 3 mM. Nonlimitingexemplary copper ion sources that produce Cu(II) ions include Cu(NO₃)₂Cu(OAc)₂ or CuSO₄, metallic Cu and metallic Cu with sonication. In someembodiments, a copper ion source that produces Cu(II) ions is includedin a click reaction at a concentration between 0.01 mM and 10 mM,between 0.01 mM and 5 mM, between 0.05 mM and 5 mM, between 0.1 mM and 5mM, between 0.5 mM and 5 mM, between 0.5 mM and 4 mM, or between 0.5 mMand 3 mM.

In some embodiments, a copper ion source is copper-containing metal,such as copper wire.

Nonlimiting exemplary reducing agents that may be used to reduce Cu(II)ions to Cu(I) ions include ascorbate, tris(2-carboxyethyl) phosphine(TCEP), NADH, NADPH, thiosulfate, metallic copper, hydroquinone, vitaminK₁, glutathione, cysteine, 2-mercaptoethanol, and dithiothreitol.Nonlimiting exemplary metals that may act as reducing agents include Al,Be, Co, Cr, Fe (including Fe²⁺), Mg, Mn, Ni, Zn, Au, Ag, Hg, Cd, Zr, Ru,Fe, Co (including Co²⁺), Pt, Pd, Ni, Rh, and W. In some embodiments, areducing agent is included in a click reaction at a concentration 1micromolar to 5 molar.

In some embodiments, a reducing agent is an applied electric potential.In this case, a ligand such as TBTA, THPTA, benxzimidazole, BCS, etc isused employed and an electric potential of −30 to −300 mV is applied ina two compartment cell using a combination of working and referenceelectrodes. Standard buffers can be used (HEPES, Tris, etc) and theelectric potential may be applied during the course of the reaction. SeeChemBioChem 2008, 9, 1481-1486. for further details and experimentalinformation.

Copper Ion Chelators

Without limitation to any specific mechanism, it is known that coppercan promote the cleavage of biomolecules, such as proteins and nucleicacids. The addition of a copper chelator in a click reaction may reducethe detrimental effects of copper, thereby preserves the structuralintegrity of biomolecules.

In some embodiments, a click reaction comprises a copper chelator. Insome embodiments, a copper chelator stabilizes Cu(I) ions againstoxidation, precipitation, and/or disproportionation. By including acopper chelator, in some embodiments, a lower concentration of copperions can be used in a click reaction to achieve the same efficiency aswould be obtained in the presence of higher concentrations of copperions in the absence of a chelator.

Nonlimiting exemplary copper ion chelators include compounds of formula(V):

wherein X, Y, and Z each independently have the formula:

wherein:R₁, R₂, R₃, R₄ and R₅ are independently selected from hydrogen, halogen,alkyl, heteroalkyl, alkoxy, cycloalkyl, heterocycloalkyl, substitutedalkyl, substituted heteroalkyl, substituted alkoxy, substitutedcycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substitutedaryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl; andL₁, L₂, and L₃ are independently selected from alkyl, heteroalkyl,substituted alkyl, and substituted heteroalkyl, having a chain length of1-5 atoms.

In some embodiments, R₁, R₂, R₃, R₄ and R₅ for at least one substituentselected from X, Y, and Z are each H. In some embodiments, L₁, L₂ and L₃are each alkyl groups having a chain length of 1-5 atoms. In someembodiments, L₁, L₂ and L₃ are each —CH₂CH₂—.

Nonlimiting exemplary copper ion chelators also include 1,10phenanthroline-containing copper (I) chelators, such as, for example,bathophenanthroline disulfonic acid (4,7-diphenyl-1,10-phenanthrolinedisulfonic acid) and bathocuproine disulfonic acid (BCS;2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate). Nonlimitingexemplary chelators also includetris-(hydroxypropyltriazolylmethyl)amine (THPTA; see, e.g., Jentzsch etal., Inorganic Chemistry, 48(2): 9593-9595 (2009)) and the Cu(I)chelators described in U.S. Publication No. US2010/0197871, thedisclosure of which is incorporated herein by reference. Nonlimitingexemplary chelators also include N-(2-acetamido)iminodiacetic acid(ADA), pyridine-2,6-dicarboxylic acid (PDA), S-carboxymethyl-L-cysteine(SCMC), trientine, tetra-ethylenepolyamine (TEPA),N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), EDTA,neocuproine, N-(2-acetamido)iminodiacetic acid (ADA),pyridine-2,6-dicarboxylic acid (PDA), S-carboxymethyl-L-cysteine (SCMC),tris-(benzyl-triazolylmethyl)amine (TBTA), and derivatives thereof. Insome embodiments, histidine is used as a chelator. In some embodiments,glutathione is used as a chelator and a reducing agent.

In some embodiments, a copper chelator, such as a compound of formula(V), is included in a click reaction at molar ratio of 1:1, 2:1, 3:1,4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or greater than 10:1, relative tothe concentration of copper in the click reaction. That is, in someembodiments, if copper is included in a click reaction at aconcentration of 2 mM, a copper chelator, such as a compound of formula(V), may be included in the click reaction at a concentration of 2 mM(1:1), 4 mM (2:1), 6 mM (3:1), etc. In some embodiments, theconcentration of a copper chelator, such as a compound of formula (V),in a click reaction is between 1 μM and 100 mM, between 10 μM and 10 mM,between 50 μM and 10 mM, or between 1 mM and 10 mM.

Compositions

In some embodiments, compositions are provided. In some embodiments, acomposition comprises a compound of any one of Formulas (I) to (XIII).In some embodiments, a composition comprises a compound of any one ofFormulas (I) to (XIII) and a modified biomolecule. In some suchembodiments, the compound of any one of Formulas (I) to (XIII) comprisesan azide and the biomolecule comprises an alkyne, such as a terminalalkyne or an activated alkyne, or a phosphine, such as atriarylphosphine. In some embodiments, the compound of any one ofFormulas (I) to (XIII) comprises an alkyne and the biomolecule comprisesan azide.

In some embodiments, a composition comprises a first compound of any oneof Formulas (I) to (XIII) and a second compound of any one of Formulas(I) to (XIII), wherein the first and second compounds of Formulas (I) to(XIII) are distinguishable from one another. For example, in someembodiments, the first compound of any one of Formulas (I) to (XIII)comprises a first reporter molecule and the second compound of any oneof Formulas (I) to (XIII) comprises a second reporter molecule, whereinthe first and second reporter molecules are detectably different. Insome embodiments, the first compound of any one of Formulas (I) to(XIII) comprises an alkyne and the second compound of any one ofFormulas (I) to (XIII) comprises an azide. In some such embodiments, thecomposition comprises a first biomolecule comprising an alkyne reactivegroup and a second biomolecule comprising an azide reactive group. Insome embodiments, a composition comprise three, four, five, or morecompounds of Formulas (I) to (XIII). In some such embodiments, thecompounds of Formulas (I) to (XIII) in a composition can each beindependently detected. That is, in some embodiments, two or more of thecompounds comprise detectably different reporter molecules and/or can beseparated from one another prior to detection, etc.

In some embodiments, a composition further comprises a copper ion sourceand/or a reducing agent and/or a copper ion chelator. In some suchembodiments, the copper ion chelator is a compound of formula (V).

Various buffering agents can be included in the compositions describedherein, including inorganic and organic buffering agents. In someembodiments buffering agent is a zwitterionic buffering agent. Exemplarybuffering agents include phosphate (such as, for example, in phosphatebuffered saline (PBS)), succinate, citrate, borate, maleate, cacodylate,N-(2-Acetamido)iminodiacetic acid (ADA), 2-(N-morpholino)-ethanesulfonicacid (MES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES),piperazine-N,N′-2-ethanesulfonic acid (PIPES),2-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),N,N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid (BES),3-(N-morpholino)-propanesulfonic acid (MOPS),N-tris-(hydroxymethyl)-2-ethanesulfonic acid (TES),N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES),3-(N-tris-(hydroxymethyl) methylamino)-2-hydroxypropanesulfonic acid(TAPSO), 3-(N,N-Bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid(DIPSO), N-(2-Hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid)(HEPPSO), 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS),N-[Tris(hydroxymethyl)methyl]glycine (Tricine),N,N-Bis(2-hydroxyethyl)glycine (Bicine),(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid(TAPS), N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonicacid (AMPSO), tris (hydroxy methyl) amino-methane (Tris),TRIS-Acetate-EDTA (TAE), glycine,bis[2-hydroxyethyl]iminotris[hydroxymethyl]methane (BisTris), andcombinations thereof. In some embodiments, a composition furthercomprises ethylene diamine tetraacetic acid (EDTA).

The concentration of such buffering agents in a composition, in someembodiments, is between 0.1 mM and 1 M, between 10 mM and 1 M, between20 mM and 500 mM, between 50 mM and 300 mM, between 0.1 mM and 50 mM,and between 0.5 mM and 20 mM.

One skilled in the art can select a suitable composition pH according tothe intended application. In order to retain the structural integrity ofbiomolecules, in some embodiments, the pH is maintained in aphysiological range, such as, for example, between about 6.5 and 8. Insome embodiments, a composition has a pH of between 5 and 9 at 25° C.,between 6 and 8.5 at 25° C., between 6 and 8 at 25° C., between 6.5 and8 at 25° C., or between 6.5 and 7.5 at 25° C.

In some embodiments, a composition comprises one or more non-ionicdetergents. Non-limiting examples of such non-ionic detergents includepolyoxyalkylene diols, ethers of fatty alcohols (such as alcoholethoxylates), alkyl phenol ethoxylates, ethylene oxide/propylene oxideblock copolymers, polyoxyethylene ester of a fatty acids, alkyl phenolsurfactants, polyoxyethylene mercaptan analogs of alcohol ethoxylates,polyoxyethylene adducts of alkyl amines, polyoxyethylene alkyl amides,sorbitan esters, and alcohol phenol ethoxylate. Non-limiting examples ofsorbitan esters include polyoxyethylene(20) sorbitan monolaurate(TWEEN20), polyoxyethylene(20) sorbitan monopalmitate (TWEEN40),polyoxyethylene(20) sorbitan monostearate (TWEEN60) andpolyoxyethylene(20) sorbitan monooleate (TWEEN 80). In some embodiments,the concentration of such non-ionic detergents in a composition isbetween 0.005 and 0.5%, between 0.01 and 0.4%, between 0.01 and 0.3%,between 0.01 and 0.2%, or between 0.01 and 0.2%.

Conjugation of Modified Biomolecules

In various embodiments, the modified biomolecules described herein maybe linked to at least one moiety selected from a reporter molecule, acarrier molecule, a solid phase, and a therapeutic molecule, byconjugating the modified biomolecule to a compound of any one ofFormulas (I) to (XIII) using a click reaction, a 1,3-dipolarcycloaddition reaction, or Staudinger ligation reaction. In someembodiments, the reaction is carried out at room temperature in aqueoussolution.

In some embodiments, a click reaction is carried out in the presence ofcopper, such as Cu(I) ions. In some embodiments, a click reaction iscarried out in the presence of a reducing agent. In some embodiments,the click reaction is carried out in the presence of a copper chelator.In some embodiments, the resulting conjugated product is stable in anaqueous environment for sufficient time to allow manipulation,quantification, and/or detection of the biomolecule.

In some embodiments, the modified biomolecule comprises an azide moiety.In some such embodiments, the compound of any one of Formulas (I) to(XIII) comprises a terminal alkyne at substituent G. In someembodiments, the modified biomolecule comprises an alkyne moiety, suchas a terminal alkyne or an activated alkyne. In some such embodiments,the compound of any one of Formulas (I) to (XIII) comprises an azide atsubstituent G. In some embodiments, the modified biomolecule comprises aphosphine moiety, such as a triarylphosphine. In some such embodiments,the compound of any one of Formulas (I) to (XIII) comprises an azide atsubstituent G.

In some embodiments, the click reaction, 1,3-dipolar cycloadditionreaction, or Staudinger ligation reaction is carried out in a cell, in acell lysate, in a solution comprising an isolated modified biomolecule,or with a modified biomolecule immobilized on a solid support.

In some embodiments, a modified biomolecule comprises more than one typeof chemical handle. As a nonlimiting example, in some embodiments, amodified biomolecule comprises an azide and an alkyne, such as aterminal alkyne or an activated alkyne. In some such embodiments, themodified biomolecule may be conjugated to a compound of any one ofFormulas (I) to (XIII) comprising a terminal alkyne and/or a compound ofany one of Formulas (I) to (XIII) comprising an azide, using clickchemistry. In some embodiments, the modified biomolecule may beconjugated to a compound of any one of Formulas (I) to (XIII) comprisingan azide using click chemistry, and may be conjugated to anothercompound that comprises a phosphine using a Staudinger ligation orcomprises an alkyne, such as a terminal alkyne or activated alkyne,using a 1,3-bipolar cycloaddition. Alternatively, in some embodiments,the modified biomolecule may be conjugated to a compound of any one ofFormulas (I) to (XIII) comprising a terminal alkyne, and may beconjugated to another compound that comprises an azide, both using clickchemistry. Numerous combinations of chemical handles and conjugatingreagents are possible, and can be selected according to the intendedapplication by one skilled in the art.

In some embodiments, a method comprises two or more conjugationreactions. In some such embodiments, two or more conjugation reactionsoccur using the same reaction chemistry (i.e., two or more occur usingclick chemistry, 1,3-dipolar cycloaddition, or Staudinger ligation). Asa nonlimiting example, a first modified biomolecule comprises an azideand a second modified biomolecule comprises an alkyne. Both modifiedbiomolecules may be conjugated using click chemistry, eithersimultaneously or sequentially. In some such embodiments, the modifiedbiomolecules are contacted with a first compound of any one of Formulas(I) to (XIII) comprising an alkyne and a second compound of any one ofFormulas (I) to (XIII) comprising an azide. The first biomoleculecomprising the azide will be conjugated to the first compound of any oneof Formulas (I) to (XIII) comprising an alkyne, and the secondbiomolecule comprising the alkyne will be conjugated to the secondcompound of any one of Formulas (I) to (XIII) comprising an azide. Insome embodiments, in order to reduce the occurrence of the firstbiomolecule conjugating to the second biomolecule, the concentration ofthe compounds of Formulas (I) to (XIII) can be controlled appropriately(e.g., such that they are in excess with respect to the concentration ofthe biomolecules) and/or the biomolecules can be spatially distinct(e.g., in different cellular compartments).

In some embodiments, two or more conjugation reactions occur usingdifferent reaction chemistry. As a nonlimiting example, a first modifiedbiomolecule comprises an azide and a second modified biomoleculecomprises a phosphine. The first biomolecule may be conjugated to afirst compound of any one of Formulas (I) to (XIII) comprising an alkyneusing click chemistry, and the second biomolecule may be conjugated to asecond compound of any one of Formulas (I) to (XIII) comprising an azideusing Staudinger ligation. The click reaction and the Staudingerligation may be carried out either simultaneously or sequentially. Insome embodiments, in order to reduce the occurrence of the firstbiomolecule conjugating to the second biomolecule, the concentration ofthe compounds of Formulas (I) to (XIII) can be controlled appropriately(e.g., such that they are in excess with respect to the concentration ofthe biomolecules) and/or the biomolecules can be spatially distinct(e.g., in different cellular compartments).

Conjugation in a Cell

In some embodiments, methods of conjugating a modified biomolecule to acompound of any one of Formulas (I) to (XIII) in a cell are provided. Insome such embodiments, the conjugated biomolecule is separated from thecell following conjugation. In some embodiments, the conjugatedbiomolecule is identified, detected, and/or quantified in the cellularenvironment following conjugation (such as, for example, in the livecell, or in a cell that has been fixed and/or permeabilized prior toidentification, detection and/or quantification of the biomolecule).

In some embodiments, a method of conjugating a modified biomolecule to acompound of any one of Formulas (I) to (XIII) in a cell comprisescontacting a cell comprising a modified biomolecule with a compound ofany one of Formulas (I) to (XIII) under conditions allowing the compoundof any one of Formulas (I) to (XIII) to come into contact with themodified biomolecule. In some embodiments, if the modified biomoleculeis located on the surface of the cell, contacting the cell with acomposition comprising the compound of any one of Formulas (I) to (XIII)allows conjugation of the modified biomolecule. In some embodiments,when the modified biomolecule is located inside the cell, the cell maybe contacted with a composition comprising the compound of any one ofFormulas (I) to (XIII) with or without prior fixing and/orpermeabilization of the cell. In some embodiments, for example when theconjugation occurs via click reaction, the cell may also be contactedwith a copper ion source, a reducing agent, and/or a copper ionchelator. Additional components, such as buffers, detergents, salts, andthe like, can also be included in the conjugation reaction. One skilledin the art can select suitable additional components depending on theapplication.

The conjugation can be performed under aerobic or anaerobic conditions,such as under nitrogen or argon gas, and can be performed for anysuitable length of time, such as, for example, from five minutes to sixhours, from 10 minutes to 3 hours, from 20 minutes to 3 hours, or from30 minutes to 2 hours. The reaction can be performed at a wide range oftemperatures, for example, between 4° C. and 50° C., between 10° C. and40° C., or between 15° C. and 30° C.

Cells may be fixed using any method, including, but not limited totreatment with 4% formaldehyde or methanol.

Cells may be permeabilized by any method, including but not limited totreatment with NP-40 buffer or 0.1% Triton buffer.

In some embodiments, a cell comprising more than one modifiedbiomolecule is contacted with more than one compound of any one ofFormulas (I) to (XIII), wherein the compounds of Formulas (I) to (XIII)are detectably different. In some such embodiments, the cell iscontacted with two or more compounds of Formulas (I) to (XIII)simultaneously or sequentially. Nonlimiting exemplary chemical handlesthat may be used in such multiplex reactions are described above.

Following conjugation, the conjugated biomolecules may be separatedand/or detected according to methods known in the art. Exemplary suchmethods are discussed herein.

In some embodiments, a method of comprises:

(a) contacting a cell comprising a modified biomolecule with a compoundof any one of Formulas (I) to (XIII) under conditions allowingconjugation of the modified biomolecule to the compound of formulacompound of any one of Formulas (I) to (XIII) to form a conjugatedbiomolecule; and(b) detecting the conjugated biomolecule.In some embodiments, the modified biomolecule comprises an azide and thecompound of any one of Formulas (I) to (XIII) comprises a terminalalkyne. In some embodiments, the modified biomolecule comprises aterminal alkyne, an activated alkyne, or a phosphine, and the compoundof any one of Formulas (I) to (XIII) comprises an azide. In someembodiments, the method comprises separating the conjugated biomolecule,before or after (b). In some embodiments, the method comprises fixingthe cell before (a). In some embodiments, the compound of any one ofFormulas (I) to (XIII) comprises a reporter molecule. In someembodiments, the compound of any one of Formulas (I) to (XIII) comprisesa fluorophore. In some embodiments, detecting comprises illuminating theconjugated biomolecule with an appropriate wavelength of light, suchthat the reporter molecule emits light, and observing the emitted light.Conjugation in Solution

In some embodiments, methods of conjugating a modified biomolecule to acompound of any one of Formulas (I) to (XIII) in solution are provided.Such solutions include, but are not limited to, cell lysates, solutionsof isolated biomolecules (in which the biomolecules are separated fromat least some of the components of cells in which the biomolecules areordinarily found), cell supernatants, liquid biological samples(described below), and the like.

In some embodiments, a method of conjugating a modified biomolecule to acompound of any one of Formulas (I) to (XIII) in solution comprisescontacting the modified biomolecule with a compound of any one ofFormulas (I) to (XIII) under conditions allowing the compound of any oneof Formulas (I) to (XIII) to react with the modified biomolecule via aclick reaction, a 1,3-dipolar cycloaddition, or a Staudinger ligation.In some embodiments, for example when the conjugation occurs via clickreaction, a copper ion source, a reducing agent, and/or a copper ionchelator may also be included in the solution. Additional components,such as buffers, detergents, salts, and the like, can also be includedin the conjugation reaction. One skilled in the art can select suitableadditional components depending on the application.

In some embodiments, more than one modified biomolecule is present insolution. In some such embodiments, more than one compound of any one ofFormulas (I) to (XIII) is also added to the solution and conjugated tothe more that one modified biomolecules. In some embodiments, two ormore compounds of Formulas (I) to (XIII) are added to the solutionsequentially or simultaneously. In some embodiments, the compounds ofFormulas (I) to (XIII) are detectably different. Nonlimiting exemplarychemical handles that may be used in such multiplex reactions aredescribed above.

The conjugation can be performed under aerobic or anaerobic conditions,such as under nitrogen or argon gas, and can be performed for anysuitable length of time, such as, for example, from five minutes to sixhours, from 10 minutes to 3 hours, from 20 minutes to 3 hours, or from30 minutes to 2 hours. The reaction can be performed at a wide range oftemperatures, for example, between 4° C. and 50° C., between 10° C. and40° C., or between 15° C. and 30° C.

Following conjugation, the conjugated biomolecules may be separatedand/or detected according to methods known in the art. Exemplary suchmethods are discussed herein.

In some embodiments, a method of comprises:

(c) contacting a modified biomolecule with a compound of any one ofFormulas (I) to (XIII) under conditions allowing conjugation of themodified biomolecule to the compound of formula compound of any one ofFormulas (I) to (XIII) to form a conjugated biomolecule; and(d) detecting the conjugated biomolecule.In some embodiments, the modified biomolecule comprises an azide and thecompound of any one of Formulas (I) to (XIII) comprises a terminalalkyne. In some embodiments, the modified biomolecule comprises aterminal alkyne, an activated alkyne, or a phosphine, and the compoundof any one of Formulas (I) to (XIII) comprises an azide. In someembodiments, the method comprises separating the conjugated biomolecule.In some embodiments, the compound of any one of Formulas (I) to (XIII)comprises a reporter molecule. In some embodiments, the compound of anyone of Formulas (I) to (XIII) comprises a fluorophore. In someembodiments, detecting comprises illuminating the conjugated biomoleculewith an appropriate wavelength of light, such that the reporter moleculeemits light, and observing the emitted light.Conjugation on a Solid Support

In some embodiments, methods of conjugating a modified biomolecule to acompound of any one of Formulas (I) to (XIII) on a solid support areprovided. Nonlimiting exemplary such solid supports include the varioussolid supports discussed herein, including, but not limited to, solidand semi-solid matrixes, such as glass, slides, arrays, silicaparticles, polymeric particles, microtiter plates and polymeric gels. Insome embodiments, the compound of any one of Formulas (I) to (XIII)comprises a solid support as a substituent. In some embodiments, themodified biomolecule is bound to a solid support.

The modified biomolecule may be bound to a solid support through anymeans. For example, in some embodiments, the modified biomolecule mayhave been adsorbed onto a solid support through non-covalentinteractions. In some embodiments, the modified biomolecule comprises amember of a binding pair, and is bound to a solid support that comprisesthe other member of the binding pair. In some embodiments, the modifiedbiomolecule has been conjugated to a solid support through a priorreaction, which may be a click reaction, 1,3-dipolar cycloaddition, aStaudinger ligation, or other type of reaction. Thus, in someembodiments, the modified biomolecule is attached to a solid supportusing a functional group other than the chemical handle used for a clickreaction, 1,3-dipolar cycloaddition, or Staudinger ligation, whereuponthe attached modified biomolecule is then conjugated to a compound ofany one of Formulas (I) to (XIII) through the chemical handle in a clickreaction, 1,3-dipolar cycloaddition, or Staudinger ligation. By way ofexample only, the modified biomolecule can be immobilized to a solidsupport using hydroxyl, carboxyl, amino, thiol, aldehyde, halogen,nitro, cyano, amido, urea, carbonate, carbamate, isocyanate, sulfone,sulfonate, sulfonamide or sulfoxide functional groups.

When conjugation of the biomolecule to a compound of any one of Formulas(I) to (XIII) occurs on a solid support, in some embodiments, thereaction is carried out in a similar composition as is used forsolution-phase conjugation.

In some embodiments, a method of comprises:

(e) contacting a modified biomolecule with a compound of any one ofFormulas (I) to (XIII) under conditions allowing conjugation of themodified biomolecule to the compound of formula compound of any one ofFormulas (I) to (XIII) to form a conjugated biomolecule, wherein themodified biomolecule or the compound of any one of Formulas (I) to(XIII) is immobilized on a solid support; and(f) detecting the conjugated biomolecule.In some embodiments, the modified biomolecule comprises an azide and thecompound of any one of Formulas (I) to (XIII) comprises a terminalalkyne. In some embodiments, the modified biomolecule comprises aterminal alkyne, an activated alkyne, or a phosphine, and the compoundof any one of Formulas (I) to (XIII) comprises an azide. In someembodiments, the method comprises separating the conjugated biomolecule.In some embodiments, the compound of any one of Formulas (I) to (XIII)comprises a reporter molecule. In some embodiments, the compound of anyone of Formulas (I) to (XIII) comprises a fluorophore. In someembodiments, detecting comprises illuminating the conjugated biomoleculewith an appropriate wavelength of light, such that the reporter moleculeemits light, and observing the emitted light.Separation of Conjugated Biomolecules

In some embodiments, a conjugated biomolecule is separated followingconjugation via a click reaction, a 1,3-dipolar cycloaddition, or aStaudinger ligation. Nonlimiting exemplary methods of separatingconjugated biomolecules include sedimentation, centrifugation, magneticattraction, chromatographic methods, and electrophoretic methods.

In some embodiments, separation of the conjugated biomolecule isfacilitated by a substituent on a compound of any one of Formulas (I) to(XIII) that has been conjugated to the biomolecule. As a nonlimitingexample, the compound of any one of Formulas (I) to (XIII) may comprisea member of a binding pair, which is then bound to the complementarymember of the binding pair to separate the conjugated biomolecule. Forexample, in some embodiments, the compound of any one of Formulas (I) to(XIII) comprises biotin such that the conjugated biomolecule may beseparated by binding to a streptavidin-containing solid support, such asstreptavidin-coated multiwell plates or streptavidin-coatedmicroparticles. As a further non-limiting example, the compound of anyone of Formulas (I) to (XIII) may comprise a microparticle (including,for example, a magnetic microparticle) as a substituent, such that theconjugated biomolecule may be separated by centrifugation (or contactwith a magnet if the microparticle is magnetic).

In some embodiments, conjugated biomolecules are separated by thin layeror column chromatography. Nonlimiting exemplary such chromatographyincludes size exclusion, ion exchange, and affinity chromatography. Insome embodiments, conjugated biomolecules are separated usingisoelectric focusing. In some embodiments, conjugated biomolecules areseparated using electrophoresis. Nonlimiting exemplary electrophoresisincludes gel electrophoresis (such as, for example, agarose gelelectrophoresis and acrylamide gel electrophoresis), capillaryelectrophoresis, capillary gel electrophoresis, and slab gelelectrophoresis. Gel electrophoresis can be denaturing or nondenaturing,and can include denaturing gel electrophoresis followed by nondenaturinggel electrophoresis (e.g., “2D” gels). The conjugated biomolecules maybe detected at any time before, during, or after separation. In someembodiments, such as when the conjugated biomolecules are separated bygel electrophoresis, the conjugated biomolecules may be detected in theseparation medium (e.g., the gel), either during or after separation.

One skilled in the art can select a suitable separation method accordingto the moieties conjugated to the conjugated biomolecule, the identityor type of biomolecule, and the particular application.

Detection of Conjugated Biomolecules

In some embodiments, the conjugated biomolecules are detected followingconjugation. In some embodiments, a reporter molecule that is asubstituent of a compound of any one of Formulas (I) to (XIII) that hasbeen conjugated to a biomolecule is used for detection. In someembodiments, a carrier molecule that is a substituent of a compound ofany one of Formulas (I) to (XIII) that has been conjugated to abiomolecule is used for detection. In some embodiments, a solid supportthat is a substituent of a compound of any one of Formulas (I) to (XIII)that has been conjugated to a biomolecule is used for detection. Thephrase “used for detection” encompasses direct or indirect detection ofthe reporter molecule, carrier molecule, or solid support. Theconjugated biomolecules may be detected by any method. Many methods ofdetection are known in the art, and some non-limiting exemplary methodswill be discussed below by way of illustration only. One skilled in theart can select a suitable detection method depending on the identityand/or properties of the reporter molecule, carrier molecule, solidsupport, biomolecule, and any other moieties associated therewith.

Detection of conjugated biomolecules may occur at any time followingconjugation, and at any time before, during, or after separation, ifsuch separation is carried out.

In some embodiments, the moieties used for detection are anyfluorophores described herein that can be used as substituents oncompounds of Formulas (I) to (XIII). Nonlimiting exemplary suchfluorophores include fluoresceins, rhodamines, TAMRA, Alexa dyes, SYPROdyes, and BODIPY dyes.

In some embodiments, a method comprises multiplexed detection ofmodified biomolecules, for example, by conjugating the modifiedbiomolecules to compounds of Formulas (I) to (XIII) comprising differentreporter molecules. In some embodiments, the conjugation reaction can becarried out such that modified biomolecule comprising particularchemical handles are conjugated to compounds of Formulas (I) to (XIII)comprising particular reporter molecules.

By way of illustration only, as a nonlimiting example, a compositioncomprising a first modified biomolecule, a second modified biomolecule,and a third modified biomolecule is provided, wherein the first modifiedbiomolecule comprises a phosphine, the second modified biomoleculecomprises an azide, and the third modified biomolecule comprises aterminal alkyne. The composition is contacted with a first compound ofany one of Formulas (I) to (XIII) comprising a first reporter moleculeand an azide moiety in the absence of Cu(I) ions. The first compoundconjugates to the first biomolecule through a Staudinger ligation. Insome embodiments, unconjugated first compound is rendered inactiveand/or removed from the composition. Thereafter, a second compound ofany one of Formulas (I) to (XIII) comprising a second reporter moleculeand a terminal alkyne, and a third compound of any one of Formulas (I)to (XIII) comprising a third reporter molecule and an azide are added tothe composition in the presence of Cu(I) ions. The second compoundconjugates to the second modified biomolecule and the third compoundconjugates to the third modified biomolecule through a click reaction.Following conjugation, each of the three conjugated biomoleculescomprises a different reporter molecule and, in some embodiments, can bedetected in a multiplex detection method.

In some embodiments, in-gel fluorescence detection allows forquantitative differential analysis of biomolecules and is amenable tomultiplexing with other protein gel stains. In some embodiments,utilizing fluorescent- and/or UV-excitable reporter molecules assubstituents of compounds of Formulas (I) to (XIII) allows for themultiplexed detection of biomolecules (such as, for example,glycoproteins, phosphoproteins, and total proteins) in the same 1-D or2-D gels.

In some embodiments, detection of modified biomolecules (such as, forexample, proteins) can be by Western blot, in which the modifiedbiomolecules are separated by gel electrophoresis and transferred to ablotting membrane. The modified biomolecules may be conjugated on theblotting membrane to a compound of any one of Formulas (I) to (XIII),and then detected. Alternatively, in some embodiments, modifiedbiomolecules that have been previously conjugated to a compound of anyone of Formulas (I) to (XIII) can be separated by gel electrophoresisand transferred to a blotting membrane, and then detected.

Another potential aspect of “in gel” detection is the total detection ofproteins in electrophoresis gels or Western blot membranes using a“universal” click chemistry, in which phenylboronic acid-containingmolecules are tethered via a linker to an azide moiety or an alkynemoiety. The phenylboronic acid stably associates with the cis-diolmoieties on glycoproteins under certain conditions. Such phenylboronicacid-containing molecules can be used, in some embodiments, to modifyglycoproteins with either azide or alkyne moieties after electrophoreticseparation. The azide or alkyne moieties can then be used to conjugatethe glycoproteins to a compound of any one of Formulas (I) to (XIII)comprising, for example, a reporter molecule, via click chemistry,activated alkyne chemistry, or Staudinger ligation. In some embodiments,the conjugated glycoproteins may then be detected, either directly orindirectly, using, for example, the reporter molecule. In someembodiments, glycoproteins of interest can then be isolated by excisingportions of the gel comprising the modified glycoproteins, and thephenylboronic acid dissociated from the glycoproteins under acidicconditions, thereby releasing the conjugated compound of any one ofFormulas (I) to (XIII) from the glycoprotein. In some embodiments, theglycoprotein may then be identified using, for example, massspectrometry.

In some embodiments, when detection comprises detecting an opticalresponse, the conjugated biomolecules may be detected at any time byillumination with a wavelength of light that results in a detectableoptical response, and observation with a means for detecting the opticalresponse. In some embodiments, such illumination is by a violet orvisible wavelength emission lamp, an arc lamp, a laser, or even sunlightor ordinary room light, wherein the wavelength of such sources overlapthe absorption spectrum of the moiety being detected, such as afluorophore or chromophore. In some embodiments, such illumination is bya violet or visible wavelength emission lamp, an arc lamp, a laser, oreven sunlight or ordinary room light, wherein a fluorescent compounddisplays intense visible absorption as well as fluorescence emission.

In some embodiments, the illumination sources include, but are notlimited to, hand-held ultraviolet lamps, mercury arc lamps, xenon lamps,argon lasers, laser diodes, blue laser diodes, and YAG lasers. Theseillumination sources are optionally integrated into laser scanners, flowcytometer, fluorescence microplate readers, standard or minifluorometers, or chromatographic detectors. The fluorescence emissionfollowing illumination is optionally detected by visual inspection, orby use of any of the following devices: CCD cameras, video cameras,photographic film, laser scanning devices, fluorometers, photodiodes,photodiode arrays, quantum counters, epifluorescence microscopes,scanning microscopes, flow cytometers, fluorescence microplate readers,or by means for amplifying the signal such as photomultiplier tubes.

In some embodiments, for example, when a sample is examined using a flowcytometer, a fluorescence microscope, or a fluorometer, the instrumentis optionally used to distinguish and/or discriminate between multiplefluorophores having detectably different optical properties. In someembodiments, when a sample is examined using a flow cytometer,examination of the sample optionally includes isolation of particleswithin the sample based on the fluorescence response by using a sortingdevice.

In certain embodiments, fluorescence is optionally quenched using eitherphysical or chemical quenching agents.

Samples

The end user will determine the choice of the sample and the way inwhich the sample is prepared. Samples that can be used with the methodsand compositions described herein include, but are not limited to, anybiological derived material or aqueous solution that contains a modifiedbiomolecule. In certain embodiments, a samples also includes material inwhich a modified biomolecule has been added. The sample that can be usedwith the methods and compositions described herein can be a biologicalfluid including, but not limited to, whole blood, plasma, serum, nasalsecretions, sputum, saliva, urine, sweat, transdermal exudates,cerebrospinal fluid, or the like. In other embodiments, the sample arebiological fluids that include tissue and cell culture medium whereinmodified biomolecule of interest has been secreted into the medium.Cells used in such cultures include, but are not limited to, prokaryoticcells and eukaryotic cells that include primary cultures andimmortalized cell lines. Such eukaryotic cells include, withoutlimitation, ovary cells, epithelial cells, circulating immune cells, βcells, hepatocytes, and neurons. In certain embodiments, the sample maybe whole organs, tissue or cells from an animal, including but notlimited to, muscle, eye, skin, gonads, lymph nodes, heart, brain, lung,liver, kidney, spleen, thymus, pancreas, solid tumors, macrophages,mammary glands, mesothelium, and the like.

Kits

In some embodiments, kits are provided, wherein the kits comprise acompound of any one of Formulas (I) to (XIII). In some embodiments, akit further comprises a copper ion source. In some embodiments, a kitfurther comprises a reducing agent. In some embodiments, a kit furthercomprises a copper ion chelator. In some embodiments, a kit furthercomprises a reagent for modifying a biomolecule. Nonlimiting exemplarysuch copper ion sources, reducing agents, copper ion chelators, andreagents for modifying biomolecules are described herein.

In some embodiments, a kit further comprises a copper ion chelator offormula (V).

A detailed description of the invention having been provided above, thefollowing examples are given for the purpose of illustrating theinvention and shall not be construed as being a limitation on the scopeof the invention or claims.

The following examples are intended to illustrate but not limit theinvention.

Example 1

FIG. 1A shows an exemplary method of preparing a reagent for making acompound of any one of Formulas (I) to (XIII). The exemplary method isas follows.

General Synthetic Methods

Chemicals were purchased from Sigma-Aldrich, Alfa Aesar, TCI America,Fisher Scientific, Adesis Inc, or EMD unless specified otherwise.Analytical thin-layer chromatography was performed using 0.25 mm silicagel 60_(F254) plates and visualized with 254 nm UV light or withbromocresol green. ¹H NMR spectra were recorded on a Bruker Avance 400MHz or a Varian Inova 500 MHz spectrometer. All samples were dissolvedin CDCl₃, CD₃OD, D₂O, or d₆-DMSO and chemical shifts (δ) are expressedin parts per million relative to residual solvent peak as an internalstandard. Abbreviations are: s, singlet; d, doublet; t, triplet; q,quartet; m, multiplet; br, broad. Coupling constants (J) are reported inhertz (Hz). Mass spectra were recorded using electrospray ionization(ESI) on an Applied Biosystems 200 QTRAP mass spectrometer or an Agilent1100 MSD ion trap mass spectrometer. Absorbance and fluorescenceproperties for selected compounds were determined on a Perkin ElmerLS50B Luminescence Spectrometer in HPLC-grade methanol.

Analytic LC-MS data were acquired using Waters 2695 Alliance HPLCcoupled to a single-quadrupole Waters Micromass ZQ mass spectrometer,and an Xterra MS C18 column (2.5 μm particle size, 4.6×50 mm dimension).The elution gradient is 5-95% acetonitrile/10 mM NH₄OAc, pH=7 over 20minutes. MS data were recorded simultaneously in negative and positiveionization modes. Preparative HPLC purification was performed usingWaters 600 HPLC equipped with Waters 996 diode array detector, Waters717 plus autosampler, and a Luna C18 column (Phenomenex; 5 μm particlesize, 4.6 mm×250 mm dimension).

Preparation of picolyl 6-(methoxycarbonyl)nicotinic acid (2)

12.6 g of pyridine-2,5-dicarboxylic acid was suspended in 150 mL ofmethanol and 4.5 g of 95% sulfuric acid was slowly added. The reactionwas heated to reflux for 2.5 h, cooled to 25° C. and the whole waspoured into 750 mL of deionized (DI) water at room temperature. A whiteprecipitate formed and the suspension was stirred for 20 minutes,filtered through a Buchner funnel with filter paper. The precipitate wasrinsed with deionized and collected. This material was dissolved in 150mL of dichloromethane heated to 35° C., washed once with 150 mL ofsaturated sodium bicarbonate solution. The two layers were separated,and 2 N HCl was added to the organic layer to until the pH was 1-2. Awhite precipitate formed and was filtered with a Buchner funnel fittedwith filter paper. The product was rinsed with deionized water,collected, and dried under vacuum to provide 4.75 g of compound 2. TLC(80:20 ACN:H₂O, uv): R_(f)=0.53 (bis-methyl ester R_(f)=0.83). ¹H NMR(400 MHz, d₆-DMSO): 9.12 (br s, 1H), 8.42 (t, 1H), 8.15 (t, 1H), 3.89(s, 3H).

Preparation of 5-(2,5-dioxopyrrolidin-1-yl) 2-methylpyridine-2,5-dicarboxylate (3)

100 mg of 2 was dissolved in 10 mL of dichloromethane at ambienttemperature. N-hydoxysuccinimide (95 mg) was added, followed by 133 mgof 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) hydrochloride and themixture stirred at room temperature for 2 hours. The mixture was thendiluted with 15 mL of chloroform, followed by the addition of 15 mL of2% HCl solution. The organic layer was successively washed once with 15mL of DI water. The organic layer was then dried with MgSO₄, filteredand 3 used directly in the next step. TLC (ethyl acetate, uv):R_(f)=0.53.

Preparation of methyl 5-((2-((tert-butoxycarbonyl)amino)ethyl)carbamoyl) picolinate (4)

Compound 3 is used directly by dissolving in 15 mL of dichloromethane.0.30 mL of N,N,-diisopropylethylamine was added at room temperaturefollowed by 119 mg of tert-butyl (2-aminoethyl)carbamate hydrochloridewas then added and the reaction was stirred for 50 minutes, at whichtime 30 mL of dichloromethane was added followed by 20 mL of 2 M Na₂CO₃.The layers were separated and the organic layer was dried with MgSO₄,filtered and concentrated to a residue which was taken up in 3 mL ofwarm methanol and loaded onto a silica gel chromatography column andeluted with ethyl acetate and methanol to provide 104 mg of 4 as a whitesolid. TLC (ethyl acetate, uv): R_(f)=0.32.). ¹H NMR (400 MHz, CDCl₃):9.15 (d, 1H), 8.30 (d, 1H), 8.14 (complex, 2H), 5.32 (br s, 1H), 4.00(s, 3H), 3.56 (m, 2H), 3.41 (m, 2H), 1.39 (s, 9H).

Preparation of tert-butyl (2-(6-(hydroxymethyl)nicotinamido)ethyl)carbamate (5)

Compound 4 (2.4 g) was dissolved in 50 mL of methanol and 567 mg ofNaBH₄ was carefully added to control gas evolution. THF (5 mL) was addedthe reaction was placed in a preheated bath at 65° C. The reaction wasstirred at this temperature for 35 minutes, at which time 6 mL of 2 MNa₂CO₃ was added dropwise followed by 6 mL of water. The mixture wasthen concentrated to ⅕ original volume and 100 mL of ethyl acetate wasadded. The layers were separated and the organic layer was dried withMgSO₄, filtered and concentrated to an oil, which was taken up indichloromethane and flashed with dichloromethane with 0.5% triethylamineand methanol to provide 0.91 g of 5 as a white solid. TLC (ethylacetate, uv): R_(f)=0.13.). ¹H NMR (400 MHz, d₆-DMSO): 8.89 (s, 1H),8.61 (s, 1H), 8.17 (d, 1H), 7.54 (d, 1H), 6.93 (m, 1H), 5.53 (s, 1H),4.61 (s, 2H), 3.29 (m, 2H), 3.12 (m, 2H), 2.51 (s, 2H), 1.7 (s, 9H).

Preparation of tert-butyl (2-(6-(azidomethyl)nicotinamido)ethyl)carbamate (6)

Compound 5 (132 mg) was dissolved in 5 mL of DMF. Sodium azide (218 mg)was added, followed by triphenylphosphine (129 mg) and carbontetrabromide (189 mg) and the reaction was stirred for 1 hour at whichtime 10 mL of 1 M Na₂CO₃ was added followed by 30 mL of ethyl acetate.The organic layer was washed twice with 15 mL of satt'd NaHCO₃ solution(each), the organic layer was dried with MgSO₄, filtered, andconcentrated to a clear oil. This material was then purified by flashchromotagraphy suing ethyl acetate as eluent. TLC (ethyl acetate, uv):R_(f)=0.30. ¹H NMR (400 MHz, CDCl₃): 9.04 (d, 1H), 8.19 (dd, 1H), 7.72(br s, 1H), 7.43 (d, 1H), 5.17 (t, 1H), 4.55 (s, 2H), 3.58 (q, 2H), 3.43(q, 2H), 1.43 (s, 9H).

FIG. 1B shows an exemplary method of preparing a compound of any one ofFormulas (I) to (XIII) from tert-butyl(2-(6-(azidomethyl)nicotinamido)ethyl) carbamate (6). The exemplarymethod is as follows.

Preparation of a representative azido-dye conjugated compound,6-(azidomethyl)-N-(2-(6,8-difluoro-7-hydroxy-2-oxo-2H-chromene-3-carboxamido)ethyl)nicotinamide(8)

Compound 6 (5.2 mg) was dissolved in 1 mL of DCM, cooled to 5° C., then1 mL of TFA was added and the mixture was stirred for 15 minutes, thenallowed to warm gradually to ambient temperature. The mixture was thenconcentrated to an orange oil and dried under vacuum for 2 hours atwhich time the mixture was taken up in 0.8 mL of DMF. 0.1 mL ofN,N,-diisopropylethylamine was added, followed by 4.9 mg of Pacific BlueSE (7) at ambient temperature. The mixture was stirred for 1 hour, thenthe reaction mixture was loaded directly onto a silica gel column andchromatographed with dichloromethane and methanol to provide a paleyellow oil. This material was then purified by preparative HPLC using agradient of 10 mM ammonium acetate and methanol to obtain the productof >99% purity by HPLC analysis. ¹H NMR (400 MHz, d₃-MeOD): 9.21 (t,1H), 8.98 (d, 1H), 8.68 (d, 1H), 8.26 (d, 1H), 7.57 (d, 1H), 7.26 (dd,1H), 4.58 (s, 2H), 3.67-3.64 (m, 4H).

Preparation of QSYp Azide

QSY picolyl azide (“QSYp Azide”) having the structure:

was prepared substantially as described above for compound (8), exceptQSY-SE (Life Technologies catalog # Q-10193):

was used in place of Pacific Blue SE (7). Other labeled azide compounds,such as AF488-pAzide, AF647-pAzide, and biotin-PEG-pAzide, where PEG isone or more repeating units of polyethylene glycol. These compounds canbe made using a substantially similar method as with compound 8.Selected examples of compounds synthesized in this manner include thefollowing structures:

N-Ethyl biotin picolyl azide: MS (ESI): MH+=475.3, 473.3 (negativemode).

AF 488-picolyl azide: LCMS (ESI): Xterra C8, 50 mm×2.1 mm at 0.2 mL/min40-100% ACN/10 mM NH₄OAc, pH=7 over 20 minutes. T_(r)=2.1 mM,MH+=737.05.TAMRA picolyl azide:

AF 647-picolyl azide: LCMS (ESI): Xterra C8, 50 mm×2.1 mm at 0.2 mL/min.40-100% ACN/10 mM NH₄OAc, pH=7 over 20 minutes. T_(r)=2.1 min,MH+=737.05.

AF 594-picolyl azide: LCMS (ESI): Xterra C8, 50 mm×2.1 mm at 0.2 mL/min0-60% ACN/10 mM NH₄OAc, pH=7 over 20 minutes. T_(r)=2.1 min, MH+=925.3.

AF 555-picolyl alkyne (this compound was prepared in analogy to compound15. AF 488-picolyl azide: LCMS (ESI): Xterra C8, 50 mm×2.1 mm at 0.2mL/min 40-100% ACN/10 mM NH₄OAc, pH=7 over 20 minutes. T_(r)=6.17 min,MH+=1048.39.

FIG. 2 shows an exemplary method of preparing a compound of any one ofFormulas (I) to (XIII). The exemplary method is as follows.

Preparation of 6-((prop-2-yn-1-yloxy)methyl)nicotinic acid (10)

Methyl 6-(hydroxymethyl)nicotinate (9, 303 mg) was dissolved in THF.0.28 mL of a 9 M solution of propargyl bromide in toluene was added tothe reaction vessel at ambient temperature. After 12 hours, the reactionmixture was diluted with 50 mL of Et₂O and washed once with 30 mL of asaturated solution of sodium bicarbonate. The organic layer was driedwith MgSO₄, filtered, and concentrated to a residue, which waschromatographed on a silica gel column with ethyl acetate and hexanes toobtain an oil. TLC (ethyl acetate, uv): R_(f)=0.64. ¹H NMR (400 MHz,CDCl₃): 9.16 (d, 1H), 8.32 (dd, 1H), 7.57 (d, 1H), 4.80 (s, 2H), 4.33(dd, 2H), 3.96 (s, 3H), 2.50 (t, 1H).

Preparation of 6-((prop-2-yn-1-yloxy)methyl)nicotinic acid (11)

Compound 10 (18 mg) was dissolved in 0.5 mL of methanol. LiOH (0.131 mLof a 2 M aqueous solution) was added to the mixture at ambienttemperature and the reaction was stirred for 30 minutes at which timethe reaction mixture was directly applied to a silica gel column andchromatographed with dichloromethane (DCM) and methanol to provide 11 asa light yellow oil. TLC (ethyl acetate, uv): R_(f)=0.10. ¹H NMR (400MHz, CDCl₃): 9.21 (s, 1H), 8.36 (d, 2H), 7.48 (s, 1H), 4.80 (s, 2H),4.31 (s, 2H), 2.49 (s, 1H).

Preparation of tert-butyl (2-(6-((prop-2-yn-1-yloxy)methyl)nicotinamido)ethyl)carbamate (12)

Compound 11 (11 mg) was dissolved in 2 mL of DCM. 0.12 mL oftriethylamine was added at ambient temperature followed by 27 μLethylchloroformate and the mixture was stirred for 2 hours, at whichtime the mixture was cooled to −15° C. and 57 mg oftert-butyl(2-aminoethyl)carbamate hydrochloride was added and themixture was stirred for 12 hours. The mixture was then applied directlyto silica gel column and chromatographed with hexanes and ethyl acetateto furnish 12 as a white powder. TLC (ethyl acetate, uv): R_(f)=0.43. ¹HNMR (400 MHz, CDCl₃): 9.02 (s, 1H), 8.17 (dd, 1H), 7.60 (br s, 1H), 7.54(d, 1H), 5.07 (br s, 1H), 4.79 (s, 2H), 4.32 (d, 2H), 3.58 (q, 2H), 3.44(m, 2H), 2.50 (t, 1H), 2.06 (s, 1H), 1.44 (s, 9H).

Preparation of Compound 15

Compound 12 (3.5 mg) was dissolved in 0.3 mL of DCM and 0.5 mL oftrifluoroacetic acid was added at 0-5° C. After 1.4 hours, the solventwas removed and the sample placed under vacuum for 3 hours. Compound 14(4.9 mg) was then added, followed by 0.60 ml of DCM, and finally 0.10 mLof N,N,-diisopropylethylamine After 2 hours, the reaction mixture wasconcentrated directly under vacuum and 0.30 mL of methanol and 0.20 mLof a solution of 8% triethylamine in water was added. The reaction wasstirred for 1 hour. 0.20 mL of a solution of 8% triethylamine in waterwas then added and the reaction was stirred for 30 minutes, thenconcentrated to a residue. This material was purified by HPLC using agradient of 10 mM NH₄OAc/MeOH. The fractions containing product werecollected and concentrated, taken up in water and lyophilized to provide15 as a red powder. Analytical HPLC: Luna C18 250 mm×4.6 mm, 5 μm columnat 1.0 mL/min with 10 mM NH₄OAc/Methanol gradient of 5-95% over 30minutes. T_(R)=20.8 minutes, >99% pure at 254 nm and 490 nm. ¹H NMR (400MHz, D₂O): 8.46 (s, 1H), 7.83 (dd, 2H), 7.74 (dd, 1H), 7.26 (s, 1H),7.08 (d, 1H), 6.60 (dd, 4H), 4.42 (s, 2H), 4.12 (d, 2H), 3.54 (m, 4H),2.80 (m, 1H), [amide NH's exchanged with D₂O].

Example 2

In order to compare the click reaction rates using a picolyl azidereactant, to a similar reactant that lacks a pyridyl group,Cu(I)-catalzyed azide-alkyne cycloaddition reactions were carried outbetween Oregon Green® 488 (“OG”) alkyne (Life Technologies, Carlsbad,Calif.) and either QSY® Azide having the structure:

or QSY® picolyl azide (“QSYp Azide;” see Example 1) having thestructure:

Each reaction contained 10 μM OG alkyne, 40 μM of QSY Azide or QSYpAzide, 5 mM sodium ascorbate, and various concentrations of CuSO₄ wereadded in a buffer containing 100 mM Tris, pH 8, and 25% 1,2 propanediol. QSY acts as a quencher of OG. Thus, the click reaction brings thedyes in close proximity, resulting in quenching of the OG fluorescentsignal.

As shown in FIG. 3, click reaction rates using picolyl reagent QSYpAzide (A) were faster than click reaction rates using reagent QSY Azide(B) at all Cu concentrations tested. Thus, the presence of the picolylmoiety increased the rate of the click reaction in that experiment.

Next, the effect of a Cu(I)-stabilizing copper chelators on the clickreactions between OG alkyne and QSY pAzide or QSY Azide was determined.In this experiment, reactions were carried out for 30 minutes in thepresence of sodium ascorbate and 2 mM, 1 mM, 0.5 mM, or 0.25 mM Cu²⁺; orin the presence of sodium ascorbate and 0.25 mM, 0.125 mM, 0.0625 mM, or0.03125 mM Cu²⁺ and THPTA having the structure:

at a molar ratio of THPTA:Cu of 0:1 to 8:1.

The results are shown in FIG. 4. As shown in that figure, at higherconcentrations of Cu²⁺ and in the absence of THPTA, very little clickreaction takes place between OG alkyne and QSY Azide in 30 minutes. Incontrast, greater than 50% of the OG alkyne is quenched in 30 minuteswhen QSY pAzide is used as a reactant, in the presence of at least 0.5mM Cu²⁺. The presence of THPTA markedly increases the extent of theclick reaction in 30 minutes for both QSY pAzide and QSY Azide (compare,for example, 0.25 mM Cu²⁺ in the presence and absence of THPTA). Thus,THPTA not only reduces the requirement for Cu²⁺ ions, which can bedetrimental to biological systems, it greatly increases the reactionrate for both picolyl and non-picolyl click reactants.

Example 3

In order to determine whether picolyl azides showed similar enhancementin click reaction rates, various alkynes were used in the followingclick reaction:

The time to completion of the reaction was determined by taking aliquotsof the reaction mixture at time points and applying sample to thin-layerchromatggraphy (TLC) plates. The TLC plates were then developed usingmixtures of organic solvents. Reaction progress was estimated by theintensity of spots corresponding to starting material and product.Reaction times are estimated.

The results of that experiment are shown below

R

Time to >2 h 10-16 min 10-16 min No reaction completion

In that experiment, use of the picolyl alkyne reactants resulted in muchshorter reaction times than the non-picolyl alkynes under the samereaction conditions.

Example 4

A fluorogenic click assay for determining the relative rate of clickreactions was used to test various azide compounds in a procedureadapted from Zhou and Fahrni, J. Am. Chem. Soc., 2004, 126, 8862-8863:

General reactions conditions: 20 μM azide, 40 μM 7-ethynyl coumarin 16,and 4 mM sodium ascorbate in 100 μM Tris buffer at pH 7.4 with 25% v/v1,2-propanediol at 25±1° C. Reactions were initiated by the addition ofCuSO4: 125 μM. Coumarin fluorescence was recorded on a Molecular DevicesSpectraMax M5 microplate reader with excitation at 320 nm and emissiondetection at 430 nm with a cutoff at 420 nm. The turn-on fluorescence ofcoumarin triazole was correlated to its concentration using acalibration curve made from known concentrations (1.25-40 μM) of thetriazole adduct between 2-picolyl azide and 7-ethynylcoumarin 16.Fluorescence measurements were taken for at least 30 min at 30 secintervals. Measurements for each azide were performed in triplicates ormore. Error was determined from the standard deviation from data sets ateach time point. Background fluorescence was subtracted from all datafor normalization.

Coumarin-alkyne 16 was prepared and characterized as previouslydescribed in Zhou and Fahrni, J. Am. Chem. Soc., 2004, 126, 8862-8863.

The various organic azide compounds used were synthesized as describedbelow.

Benzyl azide (as shown below) is commercially available:

Synthesis of 2-azidomethylpyrdine (2-picolyl azide)

This compound was prepared and characterized according to a publishedliterature procedure of Brotherton, W. S.; Michaels, H. A.; Simmons, J.T.; Clark, R. J.; Dalai, N. S.; Zhu, L. Organic Letters 2009, 11, 49544957.

Synthesis of 4-azidomethylbenzoic acid

This compound was prepared and characterized as described in Zhou andFahrni, J. Am. Chem. Soc., 2004, 126, 8862-8863.

Synthesis of 4-azidomethylnicotinic acid

Methyl 5-(azidomethyl)nicotinate (114 mg, 0.59 mmol) was dissolved inmethanol (2.5 mL). A 1.0 M solution of LiOH in water (1.78 mL, 1.78mmol) was then added and the mixture was stirred for 25 minutes, atwhich time acetic acid (60 μL) was added and the mixture was loadeddirectly onto a silica gel column equilibrated with ethyl acetate+1%acetic acid and chromatographed with ethyl acetate+1% acetic acid to 4%acetonitrile/ethyl acetate+1% acetic acid to provide 101 mg (96%) ofthis compound as a yellow solid. Rf=0.35 (ethyl acetate+1% acetic acid,254 nm UV). ¹H NMR (400 MHz, 1:1 CDCl₃:CD₃OD): 9.75 (dd, J=2.0, 0.4 Hz,1H), 8.98 (dd, J=8.0. 2.0 Hz, 1H), 8.12 (d, J=8.0 Hz, 1H), 5.35 (s, 1H),5.18 (s, 2H). MS (ESI−): 177 (M⁺, 100%; 133.0 (60%).

Synthesis of methyl-5-(azidomethyl)nicotinate

This compound was prepared and characterized as described in Khilevich,A., Liu, B., Mayhugh, D. R., Schekeryantz, J. M., and Zhang, D.Imidazolecarboxamide derivatives as mGluR2 receptor potentiators andtheir preparation, pharmaceutical compositions and use in the treatmentof depression. WO2010/009062. Jan. 21, 2010.

Synthesis of 2-azidomethyl-4-methoxypyridine

2-Hydroxymethyl-4-methoxypyridine (278 mg, 2.0 mmol) was dissolved intetrahydrofuran (15 mL) in a 50 mL round-bottomed flask under argon. Theflask was cooled to 0-5° C. with an ice/water bath for 10 minutes atwhich time, powdered KOH (157 mg, 2.8 mmol) was added followed bypara-toluenesulfonyl chloride (pTsCl). The reaction was stirred for 12hours, at which time diethyl ether (30 mL) was added. The mixture wastransferred to a separatory funnel, and a saturated solution of NaHCO₃(40 mL) was added. The organic layer was dried with MgSO₄, filtered, andconcentrated to a residue, which was chromatographed on a silica gelcolumn with a 10% to 50% gradient of ethyl acetate/hexanes. Rf=0.69(ethyl acetate, 254 nm UV). This material was then dissolved inN,N-dimethylformamide (5 mL), and sodium azide (266 mg, 4.09 mmol) wasadded and the reaction was stirred at ambient temperature for 16 hours,at which time the reaction mixture was diluted with diethyl ether (30mL) and washed with a saturated solution of NaHCO₃ (3×30 mL), then withbrine (25 mL), dried with MgSO₄, filtered and concentrated in vacuo. Theresulting residue was chromatographed over silica gel with a 15% to 50%gradient of ethyl acetate/hexanes to furnish 100 mg (30% yield) of thiscompound as a light yellow oil. Rf=0.68 (ethyl acetate, 254 nm UV). ¹HNMR (400 MHz, CDCl₃): 8.34 (d, J=5.6 Hz, 1H,), 6.81 (d, J=2.4 Hz 1H),4.39 (s, 2H), 3.81 (s, 3H).

Synthesis of 2-azidomethyl-4-chloropyridine

2-azidomethyl-4-chloropyridine was prepared and characterized asdescribed in Jung, F. H., Cephalosporin derivatives. EP Patent No.0127992. Dec. 12, 1984.

Synthesis of tert-Butyl (6-azidomethylpyridin-3-yl)carbamate

tert-Butyl (6-(hydroxymethyl)pyridin-3-yl)carbamate (24 mg, 0.107 mmol)was dissolved in DMF (2 mL) and sodium azide (35 mg, 0.54 mmol) wasadded followed by the simultaneous addition of carbon tetrabromide (179mg, 0.54 mmol) and triphenylphosphine (142 mg, 0.54 mol). The mixturewas stirred for 2 hours at ambient temperature, then diluted in ethylacetate (20 mL) and saturated NaHCO₃ solution (20 mL). The layers wereseparated, and the organic layer was dried with MgSO₄, filtered, andconcentrated to a yellow oil, which was chromatographed (15-90% ethylacetate/hexanes on silica gel) to afford this compound as a film 16.8 mg(63%). Rf=0.92 (ethyl acetate, 254 nm UV). MS (ESI+): 250.0 (M+H+, 100%;194.0, 25%).

Synthesis of 5-carbomethoxy nicotinic acid

This compound was prepared and characterized as described in Luk, K-C.,So, S-S., Zhang, J., and Zhang, Z. Preparation of oxindoles asinhibitors of MDM2-p53 interaction for the treatment of cancer.WO2006136606. Dec. 28, 2006.

Synthesis of 5-(2,5-dioxopyrrolidin-1-yl) 2-methylpyridine-2,5-dicarboxylate

5-carbomethoxy nicotinic acid (100 mg, 0.55 mmol) was dissolved indichloromethane (10 mL) at ambient temperature. N-Hydroxysuccinimide(NHS; 95 mg, 0.83 mmol) was added, followed by1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) hydrochloride (133 mg,0.69 mol) and the mixture stirred at ambient temperature for 2 hours.The mixture was then diluted with chloroform (15 mL), followed by theaddition of 2% HCl solution (15 mL). The organic layer was washed withwater (15 mL), dried with MgSO₄, and filtered. This compound was useddirectly in the next step without further purification. Rf=0.53 (ethylacetate, 254 nm UV).

Synthesis of methyl 5-(2-(tert-butoxycarbonylamino)ethyl) carbamoylpicolinate

5-(2,5-dioxopyrrolidin-1-yl) 2-methyl pyridine-2,5-dicarboxylate (155mg, 0.55 mmol) was dissolved in dichloromethane (15 mL).N,N-diisopropylethylamine (0.30 mL, 1.65 mmol) was added at ambienttemperature followed by tert-butyl (2 aminoethyl) carbamatehydrochloride (119 mg, 0.61 mmol). The reaction was stirred for 50minutes, then diluted with dichloromethane (30 mL) and 2 M Na₂CO₃solution (20 mL). The layers were separated and the organic layer wasdried with MgSO₄, filtered and concentrated in vacuo. The residue wasdissolved in warm methanol (3 mL), loaded onto a silica gelchromatography column and eluted with ethyl acetate then 95:5 ethylacetate:methanol to provide 104 mg (58%) of this compound as a whitesolid. Rf=0.32 (ethyl acetate, 254 nm UV). ¹H NMR (400 MHz, CDCl₃): 9.15(d, J=2 Hz, 1H), 8.30 (d, J=1.6 Hz, 1H), 8.14 (complex, 2H), 5.32 (br s,1H), 4.00 (s, 3H), 3.56 (m, 2H), 3.41 (m, 2H), 1.39 (s, 9H).

Synthesis tert-butyl (2-(6-(hydroxymethyl)nicotinamido)ethyl) carbamate

NaBH₄ (55 mg, 1.46 mmol) was added slowly to methyl5-(2-(tert-butoxycarbonylamino)ethyl) carbamoyl picolinate (157 mg, 0.49mmol) dissolved in methanol (3 mL). THF (10 mL) was added and thereaction flask was placed in an oil bath preheated to 65° C. Thereaction was stirred at 65° C. for 15 minutes, at which time 2 M Na₂CO₃solution (6 mL) was added over 10 minutes followed by water (6 mL). Themixture was then concentrated to ⅕ of its original volume andchromatographed directly with 0.5% triethylamine and ethylacetate/methanol to provide this compound (132 mg, 91%) as a whitesolid. Rf=0.10 (ethyl acetate, 254 nm UV). ¹H NMR (400 MHz, d6-DMSO):8.89 (s, 1H), 8.61 (s, 1H), 8.18 (d, J=8.0 Hz, 1H), 7.55 (d, J=8.0 Hz,1H), 6.93 (m, 1H), 5.53 (br s, 1H), 4.61 (s, 2H), 3.29 (m, 2H), 3.12 (d,J=6.0 Hz, 2H), 2.51 (s, 2H), 1.7 (s, 9H).

Synthesis of tert-butyl (2-(6-(azidomethyl)nicotinamido)ethyl) carbamate

tert-butyl (2-(6-(hydroxymethyl)nicotinamido)ethyl) carbamate (132 mg,0.45 mmol) was dissolved in DMF (5 mL). Sodium azide (218 mg, 3.36 mmol)was added, followed by triphenylphosphine (129 mg, 0.492 mmol) andcarbon tetrabromide (189 mg, 185 mg, 0.56 mmol). The reaction wasstirred for 1 hour at ambient temperature, at which time 2 M Na₂CO₃solution (10 mL) was added followed by ethyl acetate (30 mL). Theorganic layer was washed with saturated NaHCO₃ solution (2×15 mL), driedwith MgSO₄, filtered, and concentrated in vacuo. The resulting clear oilwas purified by silica gel chromotagraphy using ethyl acetate as eluentto furnish this compound as a white solid. Rf=0.30 (ethyl acetate, 254nm UV). ¹H NMR (400 MHz, CDCl₃): 9.04 (d, J=1.6 Hz, 1H), 8.19 (dd,J=8.1, 2.3 Hz, 1H), 7.72 (br s, 1H), 7.43 (d, J=8.1 Hz, 1H), 5.17 (t,J=8.1 Hz, 5.8 Hz, 1H), 4.55 (s, 2H), 3.58 (q, J=4.9 Hz, 2H), 3.43 (q,J=5.9 Hz, 2H), 1.43 (s, 9H). LCMS (ESI+): 296.50 (MH+, 100%), 240.38(86%); T_(R)=8.8 min.

Synthesis of tert-butyl 2-(2-(hydroxymethyl)pyridin-4 ylamino)ethylcarbamate

To a 40 mL pressure tube equipped with a stir bar was added4-chloro-2-pyridylmethanol (0.25 g, 1.74 mmol),N,N-diisopropylethylamine (1.52 mL, 8.71 mmol), and toluene (0.925 mL;8.71 mmol) was added tert-butyl (2-aminoethyl)carbamate (0.55 g, 3.50mmol). The vessel was purged with argon and then placed in an oil bathpre-heated to 125° C. The temperature of the oil bath was increased to140° C. over 30 minutes. After 2 hours, TLC (conditions below) indicatednear complete consumption of the starting material. The reaction wascooled to ambient temperature and the toluene layer was decanted.Methanol (15 mL) was added and the mixture was heated to ca. 40° C. for5 minutes to break up the solid residual. After concentrating themixture down to ⅕ of its original volume, CHCl₃ (5 mL) was added and theresidue was loaded onto a 5 cm×15 cm silica gel column equilibrated withdichoromethane (DCM)+0.5% triethylamine Flash chromatography with DCM toDCM/10% Methanol with 0.5% triethylamine provided an inseparable mixtureof the compound and starting tert-butyl (2-aminoethyl)carbamate (1:1.4molar ratio). Rf=0.38 (15% MeOH/DCM+0.1% TEA, 254 nm UV). This mixturewas used in the next step without further purification. ¹H NMR (400 MHz,CD₃OD): (9) 7.93 (d, J=5.6 Hz, 1H), 6.75 (d, J=2.4 z, 1H), 6.48 (dd,J=6.0, 2.4 Hz, 1H), 4.54 (s, 2H), 3.66 (s, 1H), 3.26 (m, 4H), 1.46 (s,9H); (tert-butyl 2-aminoethylcarbamate) 3.33 (m, 2H), 3.12 (t, J=6.4 Hz,2H), 2.71 (t, J=6.4 Hz, 2H), 1.44 (s, 9H).

Synthesis of tert-butyl 2-(2-(azidomethyl)pyridin-4-ylamino)ethylcarbamate

The mixture of tert-butyl 2-(2-(hydroxymethyl)pyridin-4-ylamino)ethylcarbamate and tert-butyl (2-aminoethyl)carbamate was dissolved inDMF (3.5 mL) in a 20 mL screw top vial. Sodium azide (91 mg, 1.40 mmol)was added followed by the simultaneous addition of triphenylphosphine(370 mg, 1.40 mmol) and carbon tetrabromide (470 mg, 1.40 mmol) atambient temperature. After 1 hour, additional sodium azide 91 mg (1.40mmol) was added followed by the simultaneous addition oftriphenylphosphine (370 mg, 1.40 mmol) and carbon tetrabromide (470 mg,1.40 mmol). The reaction was stirred for another hour and concentratedunder a stream of nitrogen to remove the solvent. The resulting residuewas chromatographed with 1% to 10% MeOH/CHCl₃ to provide this compoundas a white solid (75 mg, 27% over 2 steps). Rf=0.22 (5% MeOH/DCM+0.1%TEA, 254 nm UV). ¹H NMR (400 MHz, CD₃OD): 8.00 (d, J=6.4 Hz, 1H), 6.71(d, J=1.6 Hz, 1H), 6.63 (dd, J=6.0, 2.0 Hz, 1H), 4.38 (s, 2H), 3.33 (m,2H; overlaps with residual MeOH), 3.25 (m, 2H), 1.96 (s, 2H), 1.44 (s,9H). MS (ESI+): 293.3 (M+H+).

Synthesis of 2-azidoethylpyridine

2-azidoethylpyridine was prepared and characterized as described byBrotherton, W. S.; Michaels, H. A.; Simmons, J. T.; Clark, R. J.; Dalai,N. S.; Zhu, L. Organic Letters 2009, 11, 49544957.

Synthesis of 3-azido-1-(pyridin-2-yl)propan-1-one

2-picolinic acid (74 mg, 0.60 mmol) was dissolved in anhydrousdichloromethane (4 mL) under Argon. Triethylamine (0.84 mL, 6.0 mmol)was then added followed by ethyl chloroformate (86 μL, 0.90 mmol). Thereaction mixture was stirred for 30 minutes at ambient temperature, atwhich time 2-azidoethlamine hydrochloride (11 mg, 0.90 mmol) was added.After further stirring at ambient temperature for 12 hours, the solventwas removed in vacuo and the resulting residue was taken up in ethylacetate, loaded onto a preparatory TLC plate, and developed using 4:1hexanes:ethyl acetate solvent system. The product-containing band wascollected and rinsed with 10:1 chloroform:methanol, filtered, andconcentrated to a yellow oil (25 mg, 22%). Rf=0.64 (ethyl acetate,u.v.). ¹H NMR (400 MHz, CDCl₃): 8.76 (d, J=4 Hz, 1H), 8.14 (d, J=8 Hz,1H), 7.83 (dd, J=7.6, 6.4 Hz, 1H), 7.47 (m, 1H), 4.49 (dd, J=7.2, 6.8Hz, 2H), 3.95 (s, 1H), 1.45 (t, J=7.2 Hz, 3H).

Synthesis of 2-Azidomethylquinoline

2-Azidomethylquinoline was prepared and characterized as described byBrotherton, W. S.; Michaels, H. A.; Simmons, J. T.; Clark, R. J.; Dalai,N. S.; Zhu, L. Organic Letters 2009, 11, 4954 4957.

Synthesis of 6-bromomethyl-2,2′-bipyridine

6-bromomethyl-2,2′-bipyridine was prepared and characterized asdescribed by Murashima, T.; Tsukiyama, S.; Fujii, S.; Hayata, K.; Sakai,H.; Miyazawa, T.; Yamada, T. Organic & Biomolecular Chemistry 2005, 3,4060-4064.

Synthesis of 6-Azidomethyl-2,2′-bipyridine

To the solution of 6-bromomethyl-2,2′-bipyridine (75 mg, 0.30 mmol) inDMF (2 mL) was added sodium azide (59 mg, 0.90 mmol). The reactionmixture was stirred for 2 hours, then diluted with diethyl ether (20 mL)and saturated NaHCO₃ solution (15 mL). The layers were separated and theorganic layer was further washed with saturated NaHCO₃ solution (2×15mL), dried with MgSO₄, filtered, and concentrated to an oil. LC-MS(ESI+): 221.39 (MH+, 44%); TR=17.4 min.

Synthesis of 2-azidomethylbenzimidazole

2-azidomethylbenzimidazole was prepared and characterized as describedby Hideg, K.; Hankovszky, H. O. Synthesis-Stuttgart 1978, 313-315.

Synthesis of 5-(6-(Azidomethyl)nicotinamido)pentanoic acid

To a solution of 6-azidomethylnicotinic acid (30 mg, 0.168 mmol) inanhydrous DMF (500 μL) was added disuccinimidyl carbonate (DSC; 65 mg,0.253 mmol) and triethylamine (TEA; 120 μL, 0.840 mmol). The reactionwas allowed to proceed for 3 hours at ambient temperature. The reactionmixture was diluted with chloroform and water. Layers were separated,and the aqueous layer was extracted with chloroform three times. Thecombined organic layer was washed with brine, dried over MgSO₄, andconcentrated in vacuo. The residual mixture was purified by silicachromatography (1:1 hexanes:ethyl acetate) to afford the succinimidylester of 6-azidomethylnicotinic acid. Rf=0.67 in 9:1chloroform:methanol.

To a solution of 5-azidomethylnicotinic acid succinimidyl ester (15 mg,0.055 mmol) in anhydrous DMF (500 μL) was added 5-aminovaleric acid (32mg, 0.273 mmol) and TEA (38 μL, 0.273 mmol). The reaction proceeded for12 hours at ambient temperature. TEA and DMF were then removed in vacuo,and the resulting residue was dry-loaded in 9:1 chloroform:methanol ontoa silica column, and purified using 9:1 chloroform:methanol as eluent.Rf=0.19 in 9:1 chloroform:methanol. ¹H NMR (D₂O, 500 MHz): 8.83 (s, 1H),8.18 (d, 1H, J=8.5 Hz), 7.59 (d, 1H, J=8 Hz), 4.62 (s, 2H), 3.42 (m,2H), 2.32 (m, 2H), 1.65 (m, 4H).

Synthesis of ALEXA FLUOR-647 (AF-647)-picolyl azide conjugate

To a solution of 8-(2-aminoethyl)-6-(azidomethyl)nicotinamide (5.5 mg,0.019 mmol) in DMF (0.95 mL) was added DIPEA (100 μL) and ALEXA FLUOR647 succinimidyl ester (ALEXA FLUOR 647-SE; 20 mg, 0.016 mmol). Afterstirring at ambient temperature for 10 hours, the reaction mixture wasconcentrated and directly purified by preparative HPLC using a 30 minutegradient of 5-95% 10 mM NH4OAc/MeOH at a 1 mL/min flow rate. Fractionscontaining the product were combined and concentrated in vacuo. Theresidual was then dissolved in water (10 mL), flash-frozen, thenlyophilized to yield 13.6 mg of ALEXA FLUOR-647-picolyl azide as abright blue powder (83%). Tr=20.8 min at 647 nm. MS (ESI+): 1061.3(M+H⁺; 2%), 531.2, 6%); (ESI−): 1060.3 (Zwitterion, 17%), 540.3 (52%),529.3 (M²⁻. 100%). HPLC: >99% purity at 254 nm and 644 nm.

Synthetic Preparation of Fluorogenic Assay Reagents

To prepare the triazole adduct between 16 and, for example,2-azidomethylpyridine, 16 (20 mg, 0.073 mmol) was dissolved in DMSO (4mL). 2-azidomethylpyridine (20 mg, 0.15 mmol) was added followed by a0.50 M solution of sodium ascorbate in water (59 μL, 0.029 mmol), and a0.25 M copper(II) sulfate solution in water (30 μL, 0.007 mmol). Thereaction was stirred for 1 hour and the solvent removed in vacuo. Thecrude reaction mixture was taken up in methanol and loaded directly ontoa preparative TLC plate (0.25 mm thickness) and the plate was developedwith 95:5 acetonitrile:H2O. The product-containing silica was collectedand sonicated in chloroform (30 mL) for 3 minutes and filtered. Thefiltrate was concentrated to deliver 8.9 mg of II (30% yield) as a tansolid. Rf=0.80 (97:3 acetonitrile:H2O). ¹H NMR (400 MHz, CDCl₃): 12.27(br s, 1H), 8.58 (dd, J=4.8, 4.0 Hz, 1H), 7.92-7.82 (m, 3H), 7.44-7.35(m, 3H), 5.81 (s, 1H), 5.44 (s, 1H), 4.51 (s, 2H), 3.37 (s, 4H), 2.71(J=7.6, 6.0 Hz, 1H), 2.57 (t, J=7.6, 6.0 Hz, 1H). Excitation maxima=325nm, emission maxima=415 nm (100 mM Tris buffer with 25%1,2-propanediol). LC-MS (ESI+): 435.48 (MH+, 100%), 407.49 (65%); TR=8.4min FIG. 5B shows the results of certain click reactions in the presenceof 31 μM Cu and THPTA at a ratio of 4:1 (THPTA:Cu).

Various azides (R—N₃), including those shown below:

were reacted with 16.

Stock solutions of 16 and the azide compounds were prepared in 100 mMTris buffer containing 25% 1,2-propanediol, unless otherwise noted. Datawere recorded on a SpectraMax M5 instrument with excitation performed at320 nm and emission detection at 430 nm, with a cutoff at 420 nm. Theinstrument was set to kinetic mode. 96-well plates were used in thetop-read mode for fluorescence measurement. The total volume in eachwell was 200 μL. CuSO₄/water solutions were always added last to theplate to initiate reaction and measurements were taken at least everyminute for 60 minutes. When THPTA was included in the reaction, theTHPTA:Cu ratio was 4:1. A fixed ratio of 16:1 sodium ascorbate:Cu wasalso employed. Reagent concentrations in the stock and final solutionswere as follows:

Component Initial concentration Final concentration 16 100 μM 40 μMazides 100 μM 20 μM

The results of that experiment are shown in FIG. 5.

FIG. 5A shows the results of certain click reactions in the presence of125 μM Cu. From FIG. 5A, it can be seen that 6-(azidomethyl)nicotinicacid undergoes click reactions much faster than 4-(azidomethyl)benzoicacid. This is believed to be due to the heteroatom of6-(azidomethyl)nicotinic, which coordinates to Cu ions and in turnaccelerates the rate of reaction.

FIG. 5B shows the additive effect that ligands such as THPTA have whenadded to the reaction of picolyl azides. Click reactions are faster whena combination of epically groups and ligands are used. Even when4-azidomethylbenzoic acid is reacted with an alkyn in the presence ofTHPTA, the Click reaction is much slower than when a picolyl azide isused with our without a ligand such as THPTA.

A summary of azide heterocyclic structures and their copper(I) catalyzedazide-alkyne cycloaddition reaction conversions after 5 min and 30 minreaction times is shown below. Concentrations used were 20 μM azide, 40μM 7-ethynyl coumarin, 125 μM CuSO4, and 4 mM sodium ascorbate.Reactions were performed in 100 μM Tris pH 7.4 with 25% v/v 1,2propanediol at 25° C.

Conversion (%) Conversion (%) Azide Structure after 5 min after 30 min

<1 <1

35 80

<1 1.4

19 49

9.4 22

41 94

14 40

2.6 5.5

14 37

1.8 2.7

12 24

<1 <1

7.8 20

<1 <1

<1 <1

Several trends are apparent.

First, 2-picolyl azide and 6-(azidomethyl)nicotinic acid are much fasterreactants than their carbocyclic analogues, benzyl azide and4-(azidomethyl)benzoic acid, giving >35-fold and >19-fold improvementsin initial CuAAC rates under these conditions.

Second, among the picolyl azide structures, an azide with an electrondonating group gives a faster rate than one with electron withdrawinggroups, presumably because the former enhances the chelating strength ofpyridyl nitrogen. An exception is the picolyl azide with an alkyaminosubstituent, which gives little conversion after 30 min.

A third observation is that azido compounds with othercopper-coordinating motifs generally did not have as strong anaccelerative effect as 2-picolyl azide. 2-azidoethylpyridine, whichcontains an additional methylene spacer between the pyridyl ring and theazido group (to give a six-membered chelate ring), gave at least ˜3-foldless product after 5 min than picolyl azide.6-azidomethyl-2,2′-bipyridine and 2-(azidomethyl)benzimidazole each didnot give any product after 30 min, despite being strong copperchelators.

Example 5

The stability of GFP in the presence of various concentrations ofCu(II), Cu(II) plus sodium ascorbate (to produce Cu(I)), and Cu(II) plussodium ascorbate and THPTA, was determined as follows.

GFP (5 nM) was incubated in 100 mM Tris buffer with 25% 1,2-propanediolat 25° C. in the presence of 2 mM, 1 mM, 0.5 mM, 0.25 mM, 0.125 mM, or0.0625 mM CuSO₄. No ascorbate was included in the incubation mixtures.GFP fluorescence was then measured at various time intervals. As shownin FIG. 6A, at the highest concentration of CuSO₄, 2 mM, the GFP signalwas 60% of maximum after 30 minutes in that experiment. GFP was found tobe essentially stable at <0.5 mM Cu(II).

In the next experiment, 5 nM GFP was incubated in 100 mM Tris bufferwith 25% 1,2-propanediol at 25° C. in the presence of 2 mM, 1 mM, 0.5mM, 0.25 mM, 0.125 mM, or 0.0625 mM CuSO₄. In each mixture, sodiumascorbate was included at a concentration 10-fold greater than theCu(II) concentration (i.e., for 1 mM Cu(II), 10 mM sodium ascorbate wasincluded). GFP fluorescence was then measured at various time intervals.As shown in FIG. 6B, at the highest concentration of Cu(II) and sodiumascorbate (2 mM Cu(II) plus 20 mM sodium ascorbate), the GFP signal was53% of maximum after 38 minutes in that experiment. GFP was found to beessentially stable at <0.25 mM Cu(II) plus 2.5 mM sodium ascorbate.

Finally, GFP stability was determined in the presence of Cu(II), withand without sodium ascorbate, and with or without THPTA. In thisexperiment, 5 nM GFP was incubated in 100 mM Tris buffer with 25%1,2-propanediol at 25° C. in the presence of (i) 2 mM Cu(II); (ii) 1 mMCu(II), 10 mM sodium ascorbate; (iii) 1 mM Cu(II), 4 mM THPTA, 10 mMsodium ascorbate; or (iv) 0.125 mM Cu(II), 0.5 mM THPTA, and 10 mMsodium ascorbate. In this experiment, the mixtures were incubated for 30minutes typically. As shown in FIG. 7A, the GFP signal graduallydeclined under all of the tested conditions in that experiment. Themixtures comprising THPTA and lower concentrations of Cu(II) showed lessdegradation of the GFP signal, compared to the mixtures with Cu(II)alone or Cu(II) plus ascorbate.

To determine whether the GFP degradation would be significant during thetime frame for completing a click reaction, click reactions betweenOregon Green® (“OG”) alkyne and QSY-azide were carried under the samefour conditions (plus a copper-less control) described above. As shownin FIG. 7B, the click reactions in the presence of THPTA and low copper,which resulted in the least degradation of GFP signal in FIG. 7A, werethe fastest. At the lowest concentration of Cu(II) with THPTA (0.125 mMCu(II) plus 0.5 mM THPTA and 10 mM sodium ascorbate), the click reactionwas complete in about 20 minutes, at which time the GFP signal was stillabout 95% of maximum (see FIG. 7A). These results show thatcopper-catalyzed click reactions can be carried out in biologicalsystems without significant protein degradation.

Example 6

General Procedure for EU Labeling of RNA in Cells: A375 Cells StablyTransfected with GFP Expressing Erk2 are Plated Overnight at 5000Cells/Well Cell Density.

Human malignant melanoma (A375) cells expressing Erk2-GFP (LifeTechnologies) were cultured in L-glutamine-containing Dulbecco'smodified Eagle Medium (Life Technologies) supplemented with 10% v/vfetal bovine serum (Life Technologies), non-essential amino acids (LifeTechnologies), and 5 μg/mL blasticidin. All cells were maintained at 37°C. under 5% CO₂.

These cells are pulse with 200 μM 5-ethynyl uridine (EU) for 1 hourfollowed by fixation with 3.7% formalin in PBS for 15 minutes. The cellsare then washed twice with 3% BSA in PBS followed by permeabilizationwith 0.5% Triton in PBS for 20 minutes. The permeabilized cells werewashed twice with 3% BSA in PBS. Click reaction was carried out for 1 hrat RT followed by washing the cells twice with 3% BSA in PBS and rinsedtwice with PBS. The click conditions were combinations of with orwithout chelate and AF647-picolyl azide or AF647-azide. The clickedcells were then stained with Hoechst stain (1 ug/mL in PBS) for 30 minat RT followed by three times washing with PBS. The cells were imaged onArrayScan.

Example 7

General Procedure for HPG Labeling of Proteins in Cells.

A375 cells stably transfected with GFP expressing Erk2 are platedovernight at 5000 cells/well cell density.

These cells were pulse with with 50 μM L-homopropargylglycine (HPG) for1 h in presence or absence of 40 μM cycloheximide followed by fixationwith 3.7% formalin in PBS for 15 minutes. The cells are then washedtwice with 3% BSA in PBS followed by permeabilization with 0.5% Tritonin PBS for 20 minutes. The permeabilized cells were washed twice with 3%BSA in PBS. Click reaction was carried out for 1 hr at RT using 2 mMCuSO₄ and 10 mM Sodium Ascorbate, followed by washing the cells twicewith 3% BSA in PBS and rinsed twice with PBS. The click reactions werecarried out following combinations of chelate, Cu and AF647-picolylazide or AF647-azide.

a. 4:1 chelate/copper+picolyl azide

b. 2:1 chelate/copper+picolyl azide

c. 1:1 chelate/copper+picolyl azide

d. No chelate NO picolyl=Classic click

The clicked cells were then stained with Hoechst stain (1 ug/mL in PBS)for 30 min at RT followed by three times washing with PBS. The cellswere imaged on ArrayScan.

Example 8

General Procedure for Labeling with Phalloidin AF546 after Click HPGLabeling of Proteins in Cells.

A375 cells stably transfected with GFP expressing Erk2 are platedovernight at 5000 cells/well cell density. Prior to incubation with HPGcontaining medium, cells were washed once with DPBS with calcium andmagnesium, then grown in methionine-free DMEM (Life Technologies) for 30mM. These cells were pulsed with 50 μM HPG for 1 hr in presence orabsence of 40 μM cycloheximide followed by fixation with 3.7% formalinin PBS for 15 minutes. The cells are then washed twice with 3% BSA inPBS followed by permeabilization with 0.5% Triton in PBS for 20 minutes.The permeabilized cells were washed twice with 3% BSA in PBS. Clickreaction was carried out for 1 hr at RT using 2 mM CuSO₄ and 10 mMSodium Ascorbate, followed by washing the cells twice with 3% BSA in PBSand rinsed twice with PBS. The click reactions were carried out using4:1 chelate/copper and AF647-picolyl azide The clicked cells were thenstained with Phalloidin-AF546 (30 mM at rt) conjugate followed byHoechst stain (1 ug/mL in PBS) for 30 mM at RT followed by three timeswashing with PBS. The cells were imaged on ArrayScan.

FIGS. 13 through 17 further illustrate the generality of the use ofchelation-assisted copper (I)-catalyzed azide-alkyne cycloaddition usingpicolyl azides to label metabolically alkynes in proteins and found muchhigher detection sensitivity compared without chelation assistance.

Example 9

Monoclonal antibody anti-TSH was modified at its GlcNAc sites withUDPGalNAz using galactoyltransferase. The GalNAz groups were then clickreacted either with the standard click reaction using BCS, with DIBOclick reaction or with THPTA/Picolyl click reaction. Cu 2 mM is theoriginal Cu Click reaction with 2 mM Cu+10 mM Ascorbate for 4 mM,followed by addition of 10 mM BCS with incubation for 30 mM.THPTA/Picolyl click reactions were carried out with 0.25 mM Cu,supplemented with THPTA at a molar ratio of 0:1, 1:1, 2:1, 4:1, and 8:1for 40 minutes in the presence of 10 mM ascorbate. For all Cu containingreactions 10 μM picolyl Oregon green alkyne was used. DIBO clickreactions were carried out with DIBO-Oregon Green 488 at 20 μM for 3 h.In FIG. 18, samples of control antibodies are indicated with galT-(nogalactosyltransferase was added in the GalNAz modification step). SBP(SeeBlue® Plus 2 Pre-Stained Standard; LC5925) and M12 (Mark 12™Unstained Standard; LC5677) were used as molecular weight standards.Gels were imaged with a FUJIFILM FLA-9000 for fluorescence detection for488 nm dyes followed by SYPRO® Ruby General Protein Stain.

Example 10

Site Specific Labeling of Cell Surface Proteins with an EngineeredPicolyl Azide Ligase and Chelation-Assisted Copper(I)-CatalyzedAzide-Alkyne Cycloaddition

The picolyl azide structure was converted into a substrate (for example,5-(6-(azidomethyl)nicotinamido)pentanoic acid as shown below) for lipoicacid ligase, an Escherichia coli enzyme useful for site-specific proteinlabeling in cells. The data showed that the W37V mutant of E. colilipoic acid ligase (Lp1A) was found to efficiently catalyze the ligationof a picolyl azide derivative bearing a carboxylate terminal ontorecombinant proteins expressed on the surface of living mammalian cellsas shown in FIG. 19. The ligated picolyl azide was then chemoselectivelyderivatized with various alkyne-fluorophores, under extremely mildconditions with 50 μM copper, in high yield and with minimal observablecytotoxicity, even on living neurons. A side-by-side comparison of ourchelation-assisted copper(I)-catalyzed azide-alkyne cycloadditionprotocol for specific protein labeling on the cell surface, against ananalogous labeling protocol with alkyl azide followed bychelation-assisted copper(I)-catalyzed azide-alkyne cycloaddition showedthat the former gives a 9-fold higher signal, with minimal background,after only 5 min of labeling (for the second step).

Synthesis of 5-(6-(azidomethyl)nicotinamido)pentanoic acid

To a solution of 6-azidomethylnicotinic acid (30 mg, 0.168 mmol; fromExample 4) in anhydrous DMF (500 μL) was added disuccinimidyl carbonate(DSC; 65 mg, 0.253 mmol) and triethylamine (TEA; 120 μL, 0.840 mmol).The reaction was allowed to proceed for 3 hours at ambient temperature.The reaction mixture was diluted with chloroform and water. Layers wereseparated, and the aqueous layer was extracted with chloroform threetimes. The combined organic layer was washed with brine, dried overMgSO₄, and concentrated in vacuo. The residual mixture was purified bysilica chromatography (1:1 hexanes:ethyl acetate) to afford thesuccinimidyl ester of 6-azidomethylnicotinic acid. Rf=0.67 in 9:1chloroform:methanol.

To a solution of of 5-azidomethylnicotinic acid succinimidyl ester (15mg, 0.055 mmol) in anhydrous DMF (500 μL) was added 5-aminovaleric acid(32 mg, 0.273 mmol) and TEA (38 μL, 0.273 mmol). The reaction proceededfor 12 hours at ambient temperature. TEA and DMF were then removed invacuo, and the resulting residue was dry-loaded in 9:1chloroform:methanol onto a silica column, and purified using 9:1chloroform:methanol as eluent. Rf=0.19 in 9:1 chloroform:methanol. ¹HNMR (D₂O, 500 MHz): 8.83 (s, 1H), 8.18 (d, 1H, J=8.5 Hz), 7.59 (d, 1H,J=8 Hz), 4.62 (s, 2H), 3.42 (m, 2H), 2.32 (m, 2H), 1.65 (m, 4H).

In Vitro Lp1A-Catalyzed Picolyl Azide Ligation for Preparation of aLAP-Picolyl Azide Adduct.

The enzymatic reaction was assembled as follows: 150 μM LAP (amino acidsequence: GFEIDKVWYDLDA), 5 μM W37VLp1A, 500 μM 5-(6-(azidomethyl)nicotinamido) pentanoic acid, 1 mM ATP, and 5 mM Mg(OAc)2 in 20% v/vglycerol in Dulbecco's phosphate-buffered saline (DPBS) at 30° C. for 30min. The reaction was quenched with EDTA (final concentration 50 mM) togive the LAP-picolyl azide adduct and analyzed on a Varian Prostar HPLCusing a reverse phase C18 Microsorb-MV100 column (250×4.6 mm).Chromatograms were recorded at 210 nm. A 10-min gradient of 30-60%acetonitrile in water with 0.1% trifluoroacetic acid at a flow rate of 1mL/min was used. FIG. 20 shows these chromatograms. LAP had a retentiontime of 7.5 min; after ligation to the picolyl azide, the retention timeincreased to 11 min. The data showed that the W37V1p1A catalyzes theattachment of the picolyl azide to LAP.

Cell Culturing.

Human embryonic kidney (HEK) cells were cultured in minimal essentialmedium (MEM, Mediatech) supplemented with 10% v/v fetal bovine serum(PAA Laboratories).

For hippocompal neuron cultures, Spague Dawley rat pups were sacrificedat embryonic day 18. Hippocampal tissue was digested with papain(Worthington) and DNasel (Roche) and plated on glass coverslipspretreated with poly-D-lysine (Sigma) and mouse laminin (LifeTechnologies) in L-glutamine-containing MEM (Sigma) supplemented with10% v/v fetal bovine serum (PAA Laboratories) and B27 (LifeTechnologies). At 3 days in vitro, half of the growth medium wasreplaced with Neurobasal medium (Life Technologies) supplemented withB27 and GlutaMAX (Life Technologies).

Genetic Constructs.

Complete nucleotide sequences of the following constructs are known inthe art: Lp1A variants in pYFJ16 for expression in E. coli; LAP-CFP inpDisplay; LAP-neurexin-1β in pECFP-N1; and LAP-neuroligin-1 in pNICE.

Imaging.

Fluorescence imaging. For FIGS. 21 and 22, cells were imaged in Tyrode'sbuffer or DPBS in epifluorescence or confocal modes. For epifluorescenceimaging, we used a Zeiss AxioObserver inverted microscope with a 40×oil-immersion objective. CFP (420Y20 excitation, 425 dichroic, 475Y40emission), Alexa Fluor® 647 (630Y20 excitation, 660 dichroic, 680Y30emission) and differential interference contrast (DIC) images werecollected and analyzed using Slidebook software (Intelligent ImagingInnovations). For confocal imaging, we used a Zeiss Axiovert 200Minverted microscope with a 40× oil-immersion objective. The microscopewas equipped with a Yokogawa spinning disk confocal head, a Quad-bandnotch dichroic mirror (405Y488Y568Y647), and 491 (DPSS), 561 nm (DPSS),640 nm (DPSS) lasers (all 50 mW). YFP/Alexa Fluor® 488 (491 laserexcitation, 528Y38 emission), Alexa Fluor® 568 (561 laser excitation,617Y73 emission), Alexa Fluor® 647 (640 laser excitation, 680/30emission), and DIC images were collected using Slidebook software.Fluorescence images in each experiment were normalized to the sameintensity ranges. Acquisition times ranged from 10-1000 milliseconds.

General Protocol for Cell-Surface Protein Labeling with PRIME MethodFollowed by Chelation-Assisted Copper(I)-Catalyzed Azide-AlkyneCycloaddition.

Human embryonic kidney (HEK) cells were transfected at ˜80% confluencywith expression plasmids for LAP-tagged neurexin-1β (400 ng for a 0.95cm2 dish) and yellow fluorescent protein-tagged histone 2B protein(H2B-YFP; 100 ng) using lipofectamine 2000 (Invitrogen). 24 hr aftertransfection, cells were treated with 10 μM purified W37VLp1A, 200 μM5-(6-(azidomethyl)nicotinamido)pentanoic acid made above, 1 mM ATP, and5 mM Mg(OAc)2 in cell growth medium for 20 min at room temperature.After excess Lp1A labeling reagents had been removed by quicklyreplacing the medium 2-3 times, cells were further labeled with 20 μMAlexa Fluor® 647-alkyne, 50 μM CuSO4, 250 μM THPTA, and 2.5 mM sodiumascorbate in DPBS for 5 min at room temperature. Cells were immediatelyimaged after excess CuAAC labeling reagents were removed by 2-3 quickwashes with fresh growth medium.

FIG. 21 shows the results of chelation-assisted copper(I) catalyzedazide-alkyne cycloaddition for tagging of Alexa Fluor® 647 onto neurexinin living HEK cells. Negative controls are shown with ATP omitted(second column) or wild-type Lp1A in place of ^(W37V)Lp1A (thirdcolumn). H2B-YFP is the nuclear-localized YFP transfection marker.Negative controls are shown with ATP omitted (second column) orwild-type Lp1A in place of ^(W37V)Lp1A (third column). H2B-YFP is thenuclear-localized YFP transfection marker. Confocal images are shown.Scale bars for all images, 10 μm.

Labeling of LAP-Neuroligin-1 in Live Dissociated Neurons with PRIMEFollowed by Chelation-Assisted CuAAC.

Neurons were transfected at 5 days in vitro with expression plasmids forLAP-tagged neuroligin-1 (500 ng for a 1.9 cm2 dish) and greenfluorescent protein-tagged Homer1b (Homer-GFP; 100 ng for a 1.9 cm2dish) using Lipofectamine 2000, using half the amount of themanufacturer's recommended reagent quantity. Neurons were labeled at 11days in vitro with 10 μM purified W37VLp1A, 200 μM of 5-(6-(azidomethyl)nicotinamido)pentanoic acid made above, 1 mM ATP, and 5 mM Mg(OAc)2 inpreconditioned supplemented Neurobasal medium for 20 min at 37° C. Afterbrief rinsing in supplemented preconditioned medium, neurons werefurther labeled with 20 μM Alexa Fluor® 647-alkyne, 50 μM Tempol, 50 μMCuSO4, 250 μM THPTA, and 2.5 mM sodium ascorbate in Tyrode's buffer for5 min at room temperature. The labeling solution was then replaced withsupplemented Neurobasal medium containing 500 μM bathocuproin sulfonate,which was incubated with neurons for 30 sec. Neurons were imaged live inTyrode's buffer after 2 further washes with supplemented Neurobasalmedium.

FIG. 22 shows chelation-assisted copper(I) catalyzed azide-alkynecycloaddition for tagging of Alexa Fluor® 647 onto neuroligin-1 inliving hippocampal neurons. DIV11 (11 days in vitro) rat hippocampalneurons expressing LAP-neuroligin-1 and GFP-tagged Homer1b were labeledwith 5-(6-(azidomethyl)nicotinamido)pentanoic acid via ^(W37V)Lp1A, thenwith Alexa Fluor® 647-alkyne via chelation-assisted copper(I) catalyzedazide-alkyne cycloaddition, and imaged live after brief rinsing.Labeling conditions were the same as in the above general protocol forcell-surface protein labeling with PRIME method, except: 1) higher[CuSO₄] of 300 μM was used for the bottom row; 2) a radical scavengerTempol (50 μM) was added to the CuAAC labeling solution; and 3) abiocompatible copper chelator bathocuproine sulfonate (500 μM) was usedduring the first rinse to immediately quench the click reaction. AlexaFluor® 647 images in the second column correspond to the boxed regions 1and 2, shown at higher zoom. White arrows denote regions of focalswelling when 300 μM CuSO₄ is used. Confocal images are shown. Scalebars for all images, 10 μm.

The invention claimed is:
 1. A compound of the formula:

wherein: A is a carbon; R₁, R₂, R₄, R₅, and R₆, are hydrogen; R₃comprises X-L-, wherein: X is selected from a reporter molecule, acarrier molecule, a solid support, and a reactive group, that areoptionally bound to one or more additional fluorophores, wherein: thereporter molecule comprises a chromophore, a fluorophore, a fluorescentprotein, a phosphorescent dye, a tandem dye, a particle, a hapten, anenzyme, or a radioisotope; the carrier molecule is an amino acid, apeptide, a protein, an antibody, an antibody fragment, an antigen, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a psoralen, a hormone, a lipid, a lipidassembly, a tyramine, a synthetic polymer, a polymeric microparticle, abiological cell, a cellular compartment, an ion chelating moiety, anenzymatic substrate, or a virus; the solid support is an aerogel, ahydrogel, a resin, a silica gel, a bead, a biochip, a microfluidic chip,a silicon ship, a multi-well plate, a membrane, a polymeric membrane, aparticle, a derivatized plastic film, a glass bead, cotton, a plasticbead, alumina gel, polysaccharide, poly(acrylate), polystyrene,poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch,heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose,diazocellulose, polyvinylchloride, polypropylene, polyethylene, nylon,latex bead, a conducting metal, a nonconducting metal, glass, magneticbead, paramagnetic bead, superparamagnetic bead, or a magnetic support;the reactive group is a carboxylic acid, an activated ester ofcarboxylic acid, an amine, a hydrazine, a haloacetamide, an alkylhalide, an isothiocynate or a maleimide group; and L isNH—(CH₂)_(n)—NH—C(O)—, wherein n is 1 to 12; Z is a single covalentbond; and G is an azide.
 2. The compound of claim 1, wherein thefluorophore is a xanthene, coumarin, cyanine, pyrene, oxazine,borapolyazaindacene, or carbopyranine.
 3. The compound of claim 1,wherein the enzyme is horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or beta-lactamase.
 4. A method of modifying abiomolecule comprising the step of reacting in a solution a biomoleculecomprising an azide reactive moiety with a compound of claim 1 toprovide a modified biomolecule.
 5. The method of claim 4, wherein theazide reactive moiety comprises a terminal alkyne, an activated alkyne,or triarylphosphine.
 6. The method of claim 4, wherein the biomoleculeis a nucleic acid, oligonucleotide, protein, peptide, carbohydrate,polysaccharide, glycoprotein, lipid, hormone, drug, or prodrug.
 7. Themethod of claim 4, wherein the solution further comprises copper ions.8. The method of claim 7, wherein the method further comprises at leastone reducing agent.
 9. The method of claim 7, wherein the method furthercomprises a copper chelator.
 10. A kit comprising a compound of claim 1.11. The kit of claim 10, wherein the kit further comprises a copper ionsource.
 12. The kit of claim 10, wherein the kit further comprises atleast one reducing agent.
 13. The kit of claim 10, wherein the kitfurther comprises a copper chelator.
 14. A compound selected from thegroup consisting of: